Methods of Generating Stem Cells and Embryonic Bodies Carrying Disease-Causing Mutations and Methods of Using same for Studying Genetic Disorders

ABSTRACT

Stem cells, stem cell lines and differentiated cells, tissues and organs which carry disease-causing mutations are provided. There is also provided a method of identifying agents suitable for treating disorders associated with at least one disease-causing mutations such as myotonic dystrophy and van Waardenburg syndrome.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to human embryonic stem (ES) cells whichcarry disease-causing mutations, and more particularly, to methods ofusing such cells in developing treatment for genetic disorders such asmyotonic dystrophy and van Waardenburg syndrome.

Genetic disorders result from chromosomal aberrations such as trisomies,monosomies, deletions, duplications and inversions, and/or from DNAabnormalities such as single nucleotide substitutions, deletion,insertion, or repeat expansion in one or more genes. Such chromosomaland/or DNA abnormalities are often transmitted in a recessive (e.g.,cystic fibrosis and Canavan), dominant (e.g., Myotonic Dystrophy) orimprinting (e.g., Prader-Willi or Angelman syndromes) mode ofinheritance.

For example, myotonic dystrophy (DM1) or Steinert's disease is anautosomal dominant, late-onset, myotonic disorder affecting 2.1-14.3 outof 100,000 live-birth individuals worldwide (Meola, 2000). DM ischaracterized by progressive muscle wasting, cataract, nervous systemdysfunction, cardiac conduction abnormalities and endocrineabnormalities such as diabetes and gonadal atrophy (Mankodi andThornton, 2002). DM1 results from abnormal expansions of a (CTG)_(n)repeat in the 3′-untranslated region (3′-UTR) of the DMPK gene (GenBankAccession No. NM_(—)004409). Thus, while normal individuals exhibitbetween 5-30 repeat copies, mildly affected individuals exhibit 50-80repeat copies and severely affected individuals exhibit more than 2,000copies (Brook et al, 1992).

Other examples of autosomal dominant disorders include the VanWaardenburg syndrome (WS1, Waardenburg, 1951) and Huntington's disease(HD). Van Waardenburg syndrome is characterized by a wide bridge of thenose owing to lateral displacement of the inner canthus of each eye,pigmentary disturbance (frontal white blaze of hair, heterochromiairidis, white eye lashes, leukoderma), and cochlear deafness (McKusick1992; Waardenburg, 1951). The incidence prevalence of the disease isestimated to be between 1.44 to 2.05 newborns out of 100,000 deliveriesworldwide (Fraser, 1976). Deletion of the whole PAX3 gene (GenBankAccession No. NM 000438) or single-base substitutions in the paireddomain or the homeodomain of PAX3 were found to cause WS1 (Baldwin etal, 1992; Tassabehji et al, 1992). Huntington's disease (HD) ischaracterized by a progressive, localized neural cell death which leadsto choreic movements and dementia. The disease is associated withincreases in the length of a CAG triplet repeat present in a gene called‘huntingtin’ located on chromosome 4p16.3.

Cystic fibrosis (CF) is an autosomal recessive disorder characterized bydisruptions of the exocrine function of the pancreas, intestinal glands,biliary tree, bronchial glands, and sweat glands. CF is caused bymutations in the cystic fibrosis conductance regulator (CFTR) gene(GenBank Accession No. M28668, Kerem, B., et al., 1989, Science 245:1073-1080) and its estimated incidence in the USA is 1 out of 3419live-birth among the white population, and 1 out of 12,163 live-birthamong the other populations (Kosorok M R, et al., 1996, Stat. Med. 15:449-62).

Another example of an autosomal recessive disorder is the lysosomalstorage metachromatic leukodystrophy (MLD) disorder. MLD results frommutations in two different genes, arylsulfatase A (ARSA, GenBankAccession No. AY271820) and prosaposin (GenBank Accession No. BT006849),both of which encode for proteins needed for proper degradation ofcerebroside sulfate, a glycolipid mainly found in the myelin membranes(Gieselmann V, et al., 1994, Hum. Mutat. 4: 233-42).

Still another example of an autosomal recessive disease is spinalmuscular atrophy (SMA) which is caused by disruption of the telomericcopy of a duplicated gene called survival motor neuron (SMN1). SMA ischaracterized by degeneration of the anterior horn cells leading tosymmetrical muscle weakness and wasting of voluntary muscles.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease causedby mutation in the gene encoding dystrophin and characterized by aprogressive proximal muscular dystrophy with characteristicpseudohypertrophy of the calves. The disease affects a wide variety oftissues including, skeletal muscle, cardiac muscle, smooth muscle,nervous system, retina and myoblasts.

However, although many of such genetic disorders can be diagnosedprenatally (using chorionic villi or amniotic fluid samples), or evenprior to the implantation of an in vitro fertilized embryo (at theblastocyst stage) in the uterus, in most cases, the processes leading tothe overall disorder's phenotype are unknown.

To farther understand the molecular and physiological basis of suchdisorders and in attempts to develop proper treatments, severaldisease-models, such as cell cultures and animal models, have beenconstructed. Examples include the splotch-delayed (Spd) mouse mutantwhich carries a point mutation in the Pax-3 gene (Vogan K J, et al.,1993, Genomics. 17: 364-9; Asher et al, 1996) as a model for WS; theDMPK-deficient mice (Berul C I, et al., 2000, J. Interv. Card.Electrophysiol. 4: 351-8) and the C2C12 mouse myoblast cells expressingchimeric reporter gene fused to a human DMPK 3′-UTR (Amack J D, et al.,1999, Hum. Mol. Genet. 8: 1975-84) as models for DM1; the CF-mousemodels [e.g., delta-F508 (van Doorninck J H, et al., 1995, EMBO J. 14:4403-11) and G480C (Dickinson P et al., 2002, Mol. Genet. 11: 243-51)];and the arylsulfatase A-deficient mice (D'Hooge R, et al., 2001, BrainRes. 907: 35-43) as a model for MLD. However, although suchdisease-models present biochemical models of the disorder, they often donot reproduce the clinical symptoms (Elsea S H, Lucas R E., 2002, ILARJ. 43: 66-79), probably as a result of various cloning artifacts anddifferences in the genetic make-up between various species (i.e., mouseand human). Thus, the presently available disease-models are notsuitable for developing cures for genetic disorders.

Embryonic stem (ES) cells are pluripotent stem cells which are capableof prolonged undifferentiated proliferation while maintaining normalkaryotype, as well as differentiation into cells of all embryonic germlayers, i.e., the endoderm, ectoderm and mesoderm and developing intoall types of cells, tissues, organs and/or body parts, including a wholeorganism. Thus, ES cells may be used to study the mechanisms leading todevelopmental and differentiation processes, lineage commitment,self-maintenance and maturation of progenitor cells. Moreover, ES cellscan be used in cell-based therapy and regeneration of many genetic andacquired diseases such as Parkinson's disease, cardiac infarcts,juvenile-onset diabetes mellitus, and leukemia (Gearhart J. Science1998, 282:1061; Rossant and Nagy, Nature Biotech. 1999, 17:23).

While reducing the present invention to practice the present inventorshave uncovered that embryos carrying naturally occurring disease-causingmutations can be used to generate ES cell lines and that such ES celllines can be further differentiated to various experimental models ofthe genetic disorders associated with the disease-causing mutations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anisolated stem cell or stem cell line carrying a disease-causing mutationin a genomic polynucleotide sequence thereof.

According to another aspect of the present invention there is providedan isolated embryoid body comprising a plurality of cells at least someof which carry a disease-causing mutation in a genomic polynucleotidesequence thereof.

According to yet another aspect of the present invention there isprovided an isolated differentiated cell, tissue or organ carrying atleast one disease-causing mutation in a genomic polynucleotide sequencethereof.

According to still another aspect of the present invention there isprovided a method of identifying an agent suitable for treating adisorder associated with at least one disease-causing mutation,comprising: (a) generating a stem cell line or an embryoid body carryingthe at least one disease-causing mutation; (b) subjecting cells of thestem cell line or the embryoid body to differentiating conditions tothereby obtain differentiated cells exhibiting an effect of the at leastone disease-causing mutation and; (c) exposing the differentiated cellsto a plurality of molecules and identifying from the plurality ofmolecules at least one molecule capable of regulating the effect of theat least one disease-causing mutation on the differentiated cells, theat least one molecule being the agent suitable for treating the disorderassociated with the at least one disease-causing-mutation.

According to still further features in the described preferredembodiments the stem cell is of embryonic origin.

According to still further features in the described preferredembodiments the stem cell is of human origin.

According to still further features in the described preferredembodiments the disease-causing mutation is selected from the groupconsisting of a missense mutation, a nonsense mutation, a frameshiftmutation, a readthrough mutation, a promoter mutation, a regulatorymutation, a deletion, an insertion, an inversion, a splice mutation anda duplication.

According to still further features in the described preferredembodiments the disease-causing mutation is associated with a geneticdisorder selected from the group consisting of cystic fibrosis (CF),myotonic dystrophy (DM), van Waardenburg syndrome (WS), metachromaticleukodystrophy (OLD), Gorlin disease, Huntington's disease (HD), spinalmuscular atrophy (SMA) and Duchenne muscular dystrophy (DMD).

According to still further features in the described preferredembodiments the disease-causing mutation is selected from the groupconsisting of the W1282X as set forth in SEQ ID NO:24 associated withcystic fibrosis, the PAX3-de128 (510de128 in SEQ ID NO:34) associatedwith van Waardenburg syndrome, more than 50 (CTG) repeats as set forthin SEQ ID NO:22 associated with Myotonic dystrophy and the 1505C→T(P377L) as set forth in SEQ ID NO:21 associated with metachromaticleukodystrophy.

According to still further features in the described preferredembodiments the stem cell is capable of being maintained in anundifferentiated state for at least 41 passages.

According to still further features in the described preferredembodiments the stem cell exhibits a karyotype of 46, XX or 46, XYfollowing at least 30 passages.

According to still further features in the described preferredembodiments the stem cell exhibits pluripotent capacity following 40passages.

According to still further features in the described preferredembodiments the stem cell is suspended in a culture medium includingserum or serum replacement.

According to still further features in the described preferredembodiments the serum is provided at a concentration of at least 10% andthe serum replacement is provided at a concentration of at least 15%.

According to still further features in the described preferredembodiments the embryoid body is derived from a stem cell or a stem cellline.

According to still further features in the described preferredembodiments the embryoid body is capable of differentiating into cellsof the embryonic ectoderm, embryonic endoderm and/or embryonic mesoderm.

According to still further features in the described preferredembodiments the cells of the embryonic ectoderm are selected from thegroup consisting of neural cells, retina cells and epidermal cells.

According to still further features in the described preferredembodiments the cells of the embryonic endoderm are selected from thegroup consisting of hepatocytes, pancreatic cells and secreting cells.

According to still further features in the described preferredembodiments the cells of the embryonic mesoderm are selected from thegroup consisting of osseous cells, cartilaginous cells, elastic cells,fibrous cells, myocytes, myocardial cells, bone marrow cells,endothelial cells, smooth muscle cells, and hematopoietic cells.

According to still further features in the described preferredembodiments the embryoid body is suspended in a culture medium includingserum or serum replacement.

According to still further features in the described preferredembodiments the embryoid body is at least 1 day old.

According to still further features in the described preferredembodiments the differentiated cell is selected from the groupconsisting of neural cells, retina cells, epidermal cells, hepatocytes,pancreatic cells, osseous cells, cartilaginous cells, elastic cells,fibrous cells, myocytes, myocardial cells, bone marrow cells,endothelial cells, smooth muscle cells, and hematopoietic cells.

According to still further features in the described preferredembodiments the tissue is selected from the group consisting of braintissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone,cartilage, connective tissue, muscle tissue, cardiac tissue braintissue, vascular tissue, hematopoietic, fat tissue, renal tissue,pulmonary tissue, and gonadal tissue.

According to still further features in the described preferredembodiments the organ is selected from the group consisting of head,brain, eye, leg, hand, heart, stomach, liver kidney, lung, pancreas,ovary, and testis.

According to still further features in the described preferredembodiments the differentiated cell, tissue or organ is of human origin.

According to still further features in the described preferredembodiments the method further comprising a step of isolating lineagespecific cells from the embryoid body prior to step (b).

According to still further features in the described preferredembodiments isolating lineage specific cells is effected by sorting ofcells contained within the embryoid body via fluorescence activated cellsorter.

According to still further features in the described preferredembodiments isolating lineage specific cells is effected by a mechanicalseparation of cells, tissues and/or tissue-like structures containedwithin the embryoid body.

According to still further features in the described preferredembodiments the lineage specific cells are of the embryonic ectoderm andare selected from the group consisting of neural cells, retina cells andepidermal cells.

According to still further features in the described preferredembodiments the lineage specific cells are of the embryonic endoderm andare selected from the group consisting of hepatocytes, secretors cellsand pancreatic cells.

According to still further features in the described preferredembodiments the lineage specific cells are of the embryonic mesoderm andare selected from the group consisting of osseous cells, cartilaginouscells, elastic cells, fibrous cells, myocytes, myocardial cells, bonemarrow cells, endothelial cells, smooth muscle cells, and hematopoieticcells.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a stem cell which carry anaturally occurring disease-causing mutation.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-d are micrographs illustrating the derivation of a humanembryonic stem (ES) cell line. FIG. 1 a—an expanded blastocyst (at day6) derived from an embryo following PGD. Note that part of thetrophoectoderm layer buds as a result of the drill performed in the zonapellucida. This embryo was used for the derivation of the I-5 (WS1)line. Size bar=30 μM; FIG. 1 b—ICM outgrowth (marked by an arrow) of theI-7 (DM1) ES cell line six days post plating the whole embryo at theblastocyst stage on MEFs. Size bar=45 μM. FIG. 1 c—a colony of the I-7(DM1) cell line (at passage five) growing in the presence of MEFs. Sizebar=45 μM; FIG. 1 d—undifferentiated cells of the I-5 (SW1) ES cell lineat passage 24. Note the typical spaces between the cells. Size bar=15μM.

FIGS. 2 a-b illustrate the presence of disease-causing mutations of theVan Waardenburg syndrome (WS) and Myotonic Dystrophy (DM) in human EScell lines. FIG. 2 a—Ethidium Bromide staining of an agarose geldepicting WS-specific PCR analysis; PCR was performed using the WSspecific primers (SEQ ID NOs:5-8). Lane 1—WS-affected parent; lane2—normal individual; lane 3—I-5 (WS1) ES cell line. Note the presence oftwo PCR products in the affected parent (lane 1) and the I-5 (WS1) EScell line corresponding to the wild-type and the 28 bp-deleted alleles.FIG. 2 b—Silver staining of DM-specific PCR products. PCR was performedusing the DM specific primers (SEQ ID NOs:1-4). Lanes 1-3—PCR productsof affected individuals; lane 4—PCR products of the I-7 (DM1) ES cellline; lanes 5-6—PCR products of normal individuals. Δ=The size of repeatexpansion. Note that DM affected individuals exhibit high molecularweight bands due to an expansion of the (CTG)_(n) repeat unit by 1 kb(lane 1), 2.3 kb (lane 2) and 2.4 kb (lane 3) beyond the normal size.Also note the presence of the high molecular weight bands in the PCRproduct of the I-7 (DM1) ES cell line corresponding to expanded repeatsof 1.4 and 3.0 kb beyond the normal size of the repeat unit.

FIGS. 3 a-f are immunohistochemistry micrographs illustrating theexpression of embryonic cell surface markers on the I-5 (WS1) ES cellsfollowing 44 passages. Shown are bright (FIGS. 3 a, c, e) or dark (FIGS.3 b, d, f) field images of human I-5 (WS1) ES cells labeled withmonoclonal antibodies specific to SSEA4 (FIGS. 3 a-b), TRA-1-6 (FIGS. 3c-d), or TRA-1-81 (FIGS. 3 e-f). Size bar=50 μM.

FIGS. 4 a-f illustrate the differentiation of ES cell lines carryingdisease-causing mutations into embryoid bodies (EBs). Shown are H&Estaining of histological sections of EBs formed from the I-7 (DM1) (FIG.4 a, size bar=60 μM) or I-5 (WS1) (FIG. 4 b, size bar—30 μM) ES celllines, and representative immunohistochemistry staining ofdifferentiating cells within the EBs derived from the DM1 and WS1 EScell line using anti nestin (FIG. 4 c, WS1), insulin (FIG. 4 d, WS1) andtroponin (FIGS. 4 e and f, WS1 and DM1, respectively) antibodies. It isworth mentioning that EBs derived from both WS1 and DM1 lines expressedall of these genes, i.e., nestin, insulin and troponin. Size bar inFIGS. 4 c-f=6 μM.

FIG. 5 illustrates RT-PCR determination of the differentiation stage ofthe I-7 (DM1) or the I-5 (WS1) ES cell lines and of the embryoid bodies(EBs) derived therefrom. Lane 1—I-7 (DM1) ES cell line grown for 34passages; lane 2—the I-5 (WS1) ES cell line grown for 41 passages; lane3—five-day-old EBs derived from the I-5 (WS1) ES cell line following 40passages; lane 4—five-day-old EBs derived from the I-7 (DM1) ES cellline following 34 passages with the exception of EBs from passage 30were used as a negative control to the OCT4 expression; The specificityof the reaction was verified in the absence of RNA (lane 5).

FIGS. 6 a-d illustrate histological sections of teratomas derived fromthe I-7 (DM1) or the I-5 (WS1) ES cell lines. Teratoma sections includesecretory epithelium rich in goblet cells and stratified epithelium(FIG. 6 a, the I-5 (WS1) ESC line, size bar=60 μm), developing bonetissue containing developing bone marrow (FIG. 6 b, the I-5 (WS1) ESCline, size bar=20 μm), developing bone tissue formed (FIG. 6 c, the I-7(DM1) ESC line, size bar=30 μm) and a developing eye-like structure andepithelium (FIG. 6 d, the I-7 (DM1) ESC line, size bar=60 μm).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a human embryonic stem (ES) cells whichcarry disease-causing mutations which can be used for generatingdifferentiated cells, tissue, embryoid bodies and organs. Specifically,the present invention can be used to model genetic disorders andidentify drug molecules for the treatment of disorders such as myotonicdystrophy and van Waardenburg syndrome.

The principles and operation of the stem cells which carrydisease-causing mutations of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Genetic disorders result from chromosomal aberrations and/or DNAabnormalities which are transmitted in a recessive (e.g., cysticfibrosis and Canavan), dominant (e.g., Myotonic Dystrophy) or imprinting(e.g., Prader-Willi or Angelman syndromes) mode of inheritance.

Continuous efforts in the field of genetics, and especially, in humangenetics, resulted in various diagnostic tools for many geneticdisorders. Thus, chromosomal and DNA abnormalities can be diagnosed inaffected individuals, un-affected carriers (e.g., of a recessivedisorder) and in embryos, using chorionic villi and amniotic fluidsamples, or even prior to the implantation of an in vitro fertilizedembryo. However, for many genetic disorders, the processes leading tothe overall disorder's phenotype are still unknown.

Prior attempts to reveal the molecular and physiological basis ofgenetic disorders include the generation of several disease-models, suchas cell cultures and animal models (Vogan K J, et al., 1993, Genomics.17: 364-9; Asher et al, 1996; Berul C I, et al., 2000, J. Interv. Card.Electrophysiol. 4: 351-8; Amack J D, et al., 1999, Hum. Mol. Genet. 8:1975-84; van Doorninck J H, et al., 1995, EMBO J. 14: 4403-11; DickinsonP et al., 2002, Mol. Genet. 11: 243-51; D'Hooge R, et al., 2001, BrainRes. 907: 35-43). However, although such disease-models presentbiochemical models of the disorder, they often do not reproduce thedisorder's clinical symptoms (Elsea S H, Lucas R E., 2002, ILAR J. 43:66-79). Thus, in most cases, the presently available disease-models arenot suitable for drug development.

While reducing the present invention to practice the present inventorshave uncovered that embryos carrying naturally occurring disease-causingmutations can be used to generate ES cell lines and that such ES celllines can be further used in developing cure for genetic disorders.

As is shown in Example 1 of the Examples section which follows thepresent inventors have successfully generated ES cell lines carryingdisease-causing mutations for the van Waardenburg syndrome, MyotonicDystrophy, metachromatic leukodystrophy and cystic fibrosis.

Thus, according to one aspect of the present invention there is providedan isolated stem cell or stem cell line carrying a disease-causingmutation in a genomic polynucleotide sequence thereof.

For example, as is shown in FIGS. 2 a-b and in Example 1 of the Examplessection which follows, the I-5 and I-7 ES cell line carry the deletionof 28 bp in the Pax3 gene and abnormal (i.e., more than 50) repeats ofthe CTG trinucleotide of the DMPK, gene causing van Waardenburg syndromeand Myotonic Dystrophy, respectively.

As used herein, the phrase “stem cell” refers to a cell capable ofdifferentiating into other cell types having a particular, specializedfunction (i.e., “fully differentiated” cells) or to cells capable ofbeing maintained in an undifferentiated state, hereinafter “pluripotentstem cells” or partially differentiated state, herein “multipotent stemcells”.

The stem cell of the present invention can be an hematopoietic stem cellobtained from bone marrow tissue of an individual at any age or fromcord blood of a newborn individual, an adult tissue stem cell derivedfrom an adult tissue (e.g., adipose tissue, skin, kidney, liver,prostate, pancreas, intestine, and bone marrow), or an embryonic stem(ES) cell obtained from the embryonic tissue formed after gestation(e.g., blastocyst), or embryonic germ (EG) cells.

As is mentioned hereinabove, the stem cell of the present invention ispreferably of embryonic origin [i.e., embryonic stem (ES) or embryonicgerm (EG) cells]. ES and EG cells can differentiate into cells of allembryonic germ layers, i.e., the endoderm, ectoderm and mesoderm anddeveloping into all types of cells, tissues, organs and/or body parts,including a whole organism.

ES or EG cell carrying a disease-causing mutation can be prepared usingmethods known in the arts.

ES cells can be isolated from blastocysts which are obtained from invivo preimplantation embryos or from in vitro fertilized (IVF) embryos.Alternatively, a single cell embryo can be expanded to the blastocyststage. For the isolation of ES cells the zona pellucida is removed fromthe blastocyst, or digested using Tyrode's acidic solution (Sigma, StLouis, Mo., USA) and the inner cell mass (ICM) is isolated byimmunosurgery, in which the trophectoderm cells are lysed and removedfrom the intact ICM by gentle pipetting. The ICM is then plated in atissue culture flask containing the appropriate medium which enables itsoutgrowth. For the derivation of human ES cells, following 9 to 15 daysin culture, the ICM derived outgrowth is dissociated into clumps eitherby a mechanical dissociation or by an enzymatic degradation and thecells are then re-plated on a fresh tissue culture medium. Coloniesdemonstrating undifferentiated morphology are individually selected bymicropipette, mechanically dissociated into clumps, and re-plated.Resulting ES cells are then routinely split every 1-2 weeks. For furtherdetails on methods of preparation ES cells see Example 1 of the Examplessection which follows and Thomson et al., [U.S. Pat. No. 5,843,780;Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc.Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod 4: 706,1989] and Gardner et al., [Fertil. Steril. 69: 84, 1998].

EG cells can be prepared from the primordial germ cells. For human EGcells, the primordial germ cells are obtained from human fetuses ofabout 8-11 weeks of gestation using laboratory techniques known toanyone skilled in the arts. The genital ridges are dissociated and cutinto small chunks which are thereafter disaggregated into cells bymechanical dissociation. The EG cells are then grown in tissue cultureflasks with the appropriate medium. The cells are cultured with dailyreplacement of medium until a cell morphology consistent with EG cellsis observed, typically after 7-30 days or 1-4 passages. For additionaldetails on methods of preparation human EG cells see Shamblott et al.,[Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Pat. No.6,090,622.

ES cells can be obtained from a variety of sources including human (AmitM and Itskovitz-Eldor J., 2002, J, Anat, 200: 225), mouse (Mills A A andBradley A, 2001, Trends Genet. 17: 331-9), golden hamster [Doetschman etal., 1988, Dev Biol. 127: 224-7], rat [Iannaccone et al., 1994, DevBiol. 163: 288-92] rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130-8;Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33], severaldomestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl.43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova etal., 2001, Cloning. 3: 59-67] and non-human primate species such asRhesus monkey and marmoset (Thomson et al., 1995, Proc Natl Acad Sci U SA. 92: 7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9). The EScells are obtained from any source which can carry the genetic disorder,such a source can be an animal model of the disease or a human embryowhich naturally carries the genetic disorder. For example, ES cells canbe obtained from domestic pigs embryos carrying the G590R mutation inthe alpha1 (X) chain of type X collagen which is associated withdwarfism (Nielsen V H et al., Mamm Genome. 2000; 11: 1087-92), miceembryos carrying the 1-bp insertion (267-268 insC, codon 90 in the Cln8gene) which is associated with motor neuron degeneration (Ranta S etal., Nat Genet. 1999; 23: 233-6), feline model of mucopolysaccharidosistype VI (Nuttall J D et al., Calcif Tissue Int. 1999; 65: 47-52) andmice embryos carrying the no b-wave (nob) X-linked recessive mutation,which is a model of congenital stationary night blindness (Pardue M T etal., Invest Ophthalmol Vis Sci. 1998; 39: 2443-9). The presence of adisease-causing mutation in such ES cells can be identified usingmolecular and cytogenetic methods known in the art which are listedhereinbelow.

Although less preferred, the stem cell of the present can be anhematopoietic stem cell provided from bone marrow cells, mobilizedperipheral blood cells or cord blood cells. For example, hematopoieticstem cell can be obtained from cord blood of fetuses carrying mutationsin the IL2RG, ARTEMIS, RAG1, RAG2, ADA, CD45, JAK3, or IL7R genes whichcause severe combined immunodeficiency (SCID, Kalman L et al., GenetMed. 2004; 6: 16-26), from fetuses or adults carrying mutations in theWiskott-Aldrich syndrome (WAS) gene which are associated with congenitalthrombocytopenia (Luthi J N et al., Exp Hematol. 2003; 31: 150-8) andfrom fetuses or adults carrying the 5881G>T mutation in theerythropoietin receptor (EPOR) gene which is associated with primaryfamilial erythrocytosis (familial polycythentia, Arcasoy M O et al.,Blood. 2002; 99: 3066-9). Bone marrow cells can be obtained from thedonor by standard bone marrow aspiration techniques know in the art, forexample by aspiration of marrow from the iliac crest. Peripheral bloodstem cells are obtained after stimulation of the donor with a single orseveral doses of a suitable cytokine, such as granulocytecolony-stimulating factor (G-CSF), granulocyte/macrophagecolony-stimulating factor (GM-CSF) and interleukin-3 (IL-3). In order toharvest desirable amounts of stem cells from the peripheral blood cells,leukapheresis is performed by conventional techniques (Caspar, C. B. etal., 1993. Blood. 81: 2866-71) and the final product is tested formononuclear cells. Cord blood cells are obtained from newbornindividuals. Nucleated cells are separated from erythrocytes usingmethods known in the arts such as a bag system and separation byagglutination (see International Publication No. WO 96/17514). CD43expressing hematopoietic stem cells are enriched using combinations ofdensity centrifugation, immuno-magnetic bead purification, affinitychromatography, and fluorescent active cell sorting (FACS). CD34+enriched stem cells are then cultured in the presence of growth factorssuch as IL-3 and stem cell factor.

Alternatively and presently less preferred, the stem cell of the presentinvention can be an adult tissue stem cell which can be isolated usingmethods known in the arts [Alison, M. R., J. Pathol. 2003 200(5):547-50; Cai, J. et al., Blood Cells Mol Dis. 2003 31(1): 18-27; andCollins, A. T. et al., J Cell Sci. 2001; 114(Pt 21): 3865-72]. Forexample, adult tissue stem cells can be obtained from individuals havingsomatic mutations in the pluripotential stem cell which causesmyelodysplastic syndromes (Narayan S et al,. Pediatr Dermatol. 2001; 18:210-2).

The phrase “stem cell line” refers to a population of stem cells whichare derived from stem cells and have been maintained in culture for anextended period of time, i.e., for a time period which allows stem cellexpansion for at least 10⁶ cells.

The phrase “disease-causing mutation” refers to any chromosomal and/orDNA abnormality which is capable of causing a disease, disorder orcondition and/or an alteration in a phenotype which is associated withthe disease, disorder or condition.

The phrase “genomic polynucleotide sequence” refers to any DNA or RNApolynucleotide sequence which is derived from the stem cell or stem cellline of the present invention.

Examples for disease-causing mutations generated by chromosomalabnormalities include, but are not limited to trisomies (e.g., DownSyndrome), monosomies (e.g., Turner's syndrome), deletions (e.g.,DiGeorge syndrome), duplications (e.g., Silver-Russell syndrome),translocations (e.g., Beckwith-Wiedemann) and inversions (e.g.,Hypogonadotropic hypogonadism).

Such chromosomal abnormalities can be identified using methods known inthe arts, including chromosomal banding (e.g., G-banding, R-banding),fluorescent in situ hybridization (FISH), primed in situ labeling(PRINS), multicolor-banding (MCB) and/or quantitative FISH (Q-FISH).

Examples for disease-causing mutations generated by DNA abnormalities(e.g., single nucleotide substitution, deletion, insertion, or repeatexpansion) include, but are not limited to, a missense mutation (i.e., amutation which changes an amino acid residue in the protein with anotheramino acid residue), a nonsense mutation (i.e., a mutation whichintroduces a stop codon in a protein), a frameshift mutation (i.e., amutation, usually, deletion or insertion of nucleic acids which changesthe reading frame of the protein, and may result in an early terminationor in a longer amino acid sequence), a readthrough mutation (i.e., amutation which results in an elongated protein due to a change in acoding frame or a modified stop codon), a promoter mutation (i.e., amutation in a promoter sequence, usually 5′ to the transcription startsite of a gene, which result in up-regulation or down-regulation of aspecific gene product), a regulatory mutation (i.e., a mutation in aregion upstream or downstream, or within a gene, which affects theexpression of the gene product), a deletion (i.e., a mutation whichdeletes coding or non-coding nucleic acids in a gene sequence), aninsertion (i.e., a mutation which inserts coding or non-coding nucleicacids into a gene sequence), an inversion (i.e., a mutation whichresults in an inverted coding or non-coding sequence), a splice mutation(i.e., a mutation which results in abnormal splicing or poor splicing)and a duplication (i.e., a mutation which results in a duplicated codingor non-coding sequence).

Following is a non-limiting list of methods which can be used toidentify nucleic acid substitutions in the stem cell or stem cell lineof the present invention which result in disease-causing mutations.

Direct sequencing of a PCR product: This method is based on theamplification of a genomic sequence using specific PCR primers in a PCRreaction following by a sequencing reaction utilizing the sequence ofone of the PCR primers as a sequencing primer. Sequencing reaction canbe performed using, for example, the Applied Biosystems (Foster City,Calif.) ABI PRISM® BigDye™ Primer or BigDye™ Terminator Cycle SequencingKits.

Restriction fragment length polymorphism (RFLP): This method uses achange in a single nucleotide which modifies a recognition site for arestriction enzyme resulting in the creation or destruction of an RFLP.

For example, RFLP can be used to detect the cystic fibrosis—causingmutation, ΔF508 [deletion of a CTT at nucleotide 1653-5, GenBankAccession No. M28668, SEQ ID NO:24; Kerem B, et al., Science. 1989, 245:1073-80] in a genomic DNA derived from the stem cell or stem cell lineof the present invention. Briefly, genomic DNA is amplified using theforward [5′-GCACCATTAAAGAAAATATGAT (SEQ ID NO:25)] and the reverse[5′-CTCTTCTAGTTGGCATGCT (SEQ ID NO:26)] PCR primers, and the resultant86 or 83 bp PCR products of the wild-type or AF508 allele, respectivelyare subjected to digestion using the DpnI restriction enzyme which iscapable of differentially digesting the wild-type PCR product (resultingin a 67 and 19 bp fragments) but not the CTT-deleted allele (resultingin a 83 bp fragment).

Single nucleotide mismatches in DNA heteroduplexes are also recognizedand cleaved by some chemicals, providing an alternative strategy todetect single base substitutions, generically named the “MismatchChemical Cleavage” (MCC) (Gogos et al., Nucl. Acids Res., 18:6807-6817,1990). However, this method requires the use of osmium tetroxide andpiperidine, two highly noxious chemicals which are not suited for use ina clinical laboratory.

Allele specific oligonucleotide (ASO): In this method, anallele-specific oligonucleotide (ASO) is designed to hybridize inproximity to the polymorphic nucleotide, such that a primer extension orligation event can be used as the indicator of a match or a mis-match.Hybridization with radioactively labeled allelic specificoligonucleotides (ASO) also has been applied to the detection ofspecific SNPs (Conner et al., Proc. Natl. Acad. Sci., 80:278-282, 1983).The method is based on the differences in the melting temperature ofshort DNA fragments differing by a single nucleotide. Stringenthybridization and washing conditions can differentiate between mutantand wild-type alleles.

It will be appreciated that ASO can be applied on a PCR productgenerated from genomic DNA. For example, to detect the A455E mutation(C1496→A in SEQ ID NO:24) which causes cystic fibrosis, genomic DNA (ofthe stem cell or stem cell line of the present invention) is amplifiedusing the 5′-TAATGGATCATGGGCCATGT (SEQ ID NO:27) and the5′-ACAGTGTTGAATGTGGTGCA (SEQ ID NO:28) PCR primers, and the resultantPCR product is subjected to an ASO hybridization using the followingoligonucleotide probe: 5′-GTTGTTGGAGGTTGCT (SEQ ID NO:29) which iscapable of hybridizing to the thymidine nucleotide at position 1496 ofSEQ ID NO:1. As a control for the hybridization, the 5′-GTTGTTGGCGGTTGCT(SEQ ID NO:30) oligonucleotide probe is applied to detect the presenceof the wild-type allele essentially as described in Kerem B, et al.,1990, Proc. Natl. Acad. Sci. USA, 87:8447-8451).

Allele-specific PCR—In this method the presence of a single nucleic acidsubstitution is detected using differential extension of a mutant and/orwild-type—specific primer on one hand, and a common primer on the otherhand. For example, the detection of the cystic fibrosis Q493X mutation(C1609→T in SEQ ID NO:24) is performed by amplifying genomic DNA(derived from the stem cell or stem cell line of the present invention)using the following three primers: the common primer (i.e., will amplifyin any case): 5′-GCAGAGTACCTGAAACAGGA (SEQ ID NO:31); the wild-typeprimer (i.e., will amplify only the cytosine-containing wild-typeallele): 5′-GGCATAATCCAGGAAAACTG (SEQ ID NO:32); and the mutant primer(i.e., will amplify only the thymidine-containing mutant allele):5′-GGCATAATCCAGGAAAACTA (SEQ ID NO:33), essentially as described inKerem, 1990 (Supra).

Methylation-specific PCR (MSPCR)—This method is used to detect specificchanges in DNA methylation which are associated with imprintingdisorders such Angelman or Prader-Willi syndromes. Briefly, the DNA istreated with sodium bisulfite which converts the unmethylated, but notthe methylated, cytosine residues to uracil. Following sodium bisulfitetreatment the DNA is subjected to a PCR reaction using primers which cananneal to either the uracil nucleotide-containing allele or the cytosinenucleotide-containing allele as described in Buller A., et al., 2000,Mol. Diagn.5: 239-43.

Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE): Twoother methods rely on detecting changes in electrophoretic mobility inresponse to minor sequence changes. One of these methods, termed“Denaturing Gradient Gel Electrophoresis” (DGGE) is based on theobservation that slightly different sequences will display differentpatterns of local melting when electrophoretically resolved on agradient gel. In this manner, variants can be distinguished, asdifferences in melting properties of homoduplexes versus heteroduplexesdiffering in a single nucleotide can detect the presence of a singlenucleotide substitution (i.e., the disease-causing mutation of thepresent invention) in the target sequences because of the correspondingchanges in their electrophoretic mobilities. The fragments to beanalyzed, usually PCR products, are “clamped” at one end by a longstretch of G-C base pairs (30-80) to allow complete denaturation of thesequence of interest without complete dissociation of the strands. Theattachment of a GC “clamp” to the DNA fragments increases the fractionof mutations that can be recognized by DGGE (Abrams et al., Genomics7:463-475, 1990). Attaching a GC clamp to one primer is critical toensure that the amplified sequence has a low dissociation temperature(Sheffield et al., Proc. Natl. Acad. Sci., 86:232-236, 1989; and Lermanand Silverstein, Meth. Enzymol., 155:482-501, 1987). Modifications ofthe technique have been developed, using temperature gradients (Wartellet al., Nucl. Acids Res., 18:2699-2701, 1990), and the method can bealso applied to RNA:RNA duplexes (Smith et al., Genomics 3:217-223,1988).

Limitations on the utility of DGGE include the requirement that thedenaturing conditions must be optimized for each type of DNA to betested. Furthermore, the method requires specialized equipment toprepare the gels and maintain the needed high temperatures duringelectrophoresis. The expense associated with the synthesis of theclamping tail on one oligonucleotide for each sequence to be tested isalso a major consideration. In addition, long running times are requiredfor DGGE. The long running time of DGGE was shortened in a modificationof DGGE called constant denaturant gel electrophoresis (CDGE) (Borrensenet al., Proc. Natl. Acad. Sci. USA 88:8405, 1991). CDGE requires thatgels be performed under different denaturant conditions in order toreach high efficiency for the detection of SNPs.

A technique analogous to DGGE, termed temperature gradient gelelectrophoresis (TGGE), uses a thermal gradient rather than a chemicaldenaturant gradient (Scholz, et al., Hum. Mol. Genet. 2:2155, 1993).TGGE requires the use of specialized equipment which can generate atemperature gradient perpendicularly oriented relative to the electricalfield. TGGE can detect mutations in relatively small fragments of DNAtherefore scanning of large gene segments requires the use of multiplePCR products prior to running the gel.

Single-Strand Conformation Polymorphism (SSCP): Another common method,called “Single-Strand Conformation Polymorphism” (SSCP) was developed byHayashi, Sekya and colleagues (reviewed by Hayashi, PCR Meth. Appl.,1:34-38, 1991) and is based on the observation that single strands ofnucleic acid can take on characteristic conformations in non-denaturingconditions, and these conformations influence electrophoretic mobility.The complementary strands assume sufficiently different structures thatone strand may be resolved from the other. Changes in sequences withinthe fragment will also change the conformation, consequently alteringthe mobility and allowing this to be used as an assay for sequencevariations (Orita, et al., Genomics 5:874-879, 1989).

The SSCP process involves denaturing a DNA segment (e.g., a PCR product)that is labeled on both strands, followed by slow electrophoreticseparation on a non-denaturing polyacrylamide gel, so thatintra-molecular interactions can form and not be disturbed during therun. This technique is extremely sensitive to variations in gelcomposition and temperature. A serious limitation of this method is therelative difficulty encountered in comparing data generated in differentlaboratories, under apparently similar conditions.

Dideoxy fingerprinting (ddF): The dideoxy fingerprinting (ddF) isanother technique developed to scan genes for the presence of mutations(Liu and Sommer, PCR Methods Appli., 4:97, 1994). The ddF techniquecombines components of Sanger dideoxy sequencing with SSCP. A dideoxysequencing reaction is performed using one dideoxy terminator and thenthe reaction products are electrophoresed on nondenaturingpolyacrylamide gels to detect alterations in mobility of the terminationsegments as in SSCP analysis. While ddF is an improvement over SSCP interms of increased sensitivity, ddF requires the use of expensivedideoxynucleotides and this technique is still limited to the analysisof fragments of the size suitable for SSCP (i.e., fragments of 200-300bases for optimal detection of mutations).

In addition to the above limitations, all of these methods are limitedas to the size of the nucleic acid fragment that can be analyzed. Forthe direct sequencing approach, sequences of greater than 600 base pairsrequire cloning, with the consequent delays and expense of eitherdeletion sub-cloning or primer walling, in order to cover the entirefragment. SSCP and DGGE have even more severe size limitations. Becauseof reduced sensitivity to sequence changes, these methods are notconsidered suitable for larger fragments. Although SSCP is reportedlyable to detect 90% of single-base substitutions within a 200 base-pairfragment, the detection drops to less than 50% for 400 base pairfragments. Similarly, the sensitivity of DGGE decreases as the length ofthe fragment reaches 500 base-pairs. The ddF technique, as a combinationof direct sequencing and SSCP, is also limited by the relatively smallsize of the DNA that can be screened.

Pyrosequencing™ analysis (Pyrosequencing, Inc. Westborough, Mass., USA):This technique is based on the hybridization of a sequencing primer to asingle stranded, PCR-amplified, DNA template in the presence of DNApolymerase, ATP sulfurylase, luciferase and apyrase enzymes and theadenosine 5′ phosphosulfate (APS) and luciferin substrates. In thesecond step the first of four deoxynucleotide triphosphates (dNTP) isadded to the reaction and the DNA polymerase catalyzes the incorporationof the deoxynucleotide triphosphate into the DNA strand, if it iscomplementary to the base in the template strand. Each incorporationevent is accompanied by release of pyrophosphate (PPi) in a quantityequimolar to the amount of incorporated nucleotide. In the last step theATP sulfurylase quantitatively converts PPi to ATP in the presence ofadenosine 5′ phosphosulfate. This ATP drives the luciferase-mediatedconversion of luciferin to oxyluciferin that generates visible light inamounts that are proportional to the amount of ATP. The light producedin the luciferase-catalyzed reaction is detected by a charge coupleddevice (CCD) camera and seen as a peak in a pyrogram™. Each light signalis proportional to the number of nucleotides incorporated.

Acycloprime™ analysis (Perkin Elmer, Boston, Mass., USA): This techniqueis based on fluorescent polarization (FP) detection. Following PCRamplification of the sequence containing the SNP of interest, excessprimer and dNTPs are removed through incubation with shrimp alkalinephosphatase (SAP) and exonuclease I. Once the enzymes are heatinactivated, the Acycloprime-FP process uses a thermostable polymeraseto add one of two fluorescent terminators to a primer that endsimmediately upstream of the site of the single nucleotide substitution.The terminator(s) added are identified by their increased FP andrepresent the allele(s) present in the original DNA sample. TheAcycloprime process uses AcycloPol™, a novel mutant thermostablepolymerase from the Archeon family, and a pair of AcycloTerminators™labeled with R110 and TAMRA, representing the possible alleles for theSNP of interest. AcycloTerminator™ non-nucleotide analogs arebiologically active with a variety of DNA polymerases. Similarly to2′,3′-dideoxynucleotide-5′-triphosphates, the acyclic analogs functionas chain terminators. The analog is incorporated by the DNA polymerasein a base-specific manner onto the 3′-end of the DNA chain, and sincethere is no 3′-hydroxyl, is unable to function in further chainelongation. It has been found that AcycloPol has a higher affinity andspecificity for derivatized AcycloTerminators than various Taq mutanthave for derivatized 2′,3′-dideoxynucleotide terminators.

Reverse dot blot: This technique uses labeled sequence specificoligonucleotide probes and unlabeled nucleic acid samples. Activatedprimary amine-conjugated oligonucleotides are covalently attached tocarboxylated nylon membranes. After hybridization and washing, thelabeled probe, or a labeled fragment of the probe, can be released usingoligomer restriction, i.e., the digestion of the duplex hybrid with arestriction enzyme. Circular spots or lines are visualizedcolorimetrically after hybridization through the use of streptavidinhorseradish peroxidase incubation followed by development usingtetramethylbenzidine and hydrogen peroxide, or via chemiluminescenceafter incubation with avidin alkaline phosphatase conjugate and aluminous substrate susceptible to enzyme activation, such as CSPD,followed by exposure to x-ray film.

It will be appreciated that the disease-causing mutation of the presentinvention can be identified using various advanced single nucleotidepolymorphism (SNP) genotyping techniques, such as dynamicallele-specific hybridization (DASH, Howell, W. M. et al., 1999. Dynamicallele-specific hybridization (DASH). Nat. Biotechnol. 17: 87-8),microplate array diagonal gel electrophoresis [MADGE, Day, I. N. et al.,1995. High-throughput genotyping using horizontal polyacrylamide gelswith wells arranged for microplate array diagonal gel electrophoresis(MADGE). Biotechniques. 19: 830-5], the TaqMan system (Holland, P. M. etal., 1991. Detection of specific polymerase chain reaction product byutilizing the 5′→3′ exonuclease activity of Thermus aquaticus DNApolymerase. Proc Natl Acad Sci U S A. 88: 7276-80), as well as variousDNA “chip” technologies such as the GeneChip microarrays (e.g.,Affymetrix SNP chips) which are disclosed in U.S. patent applicationSer. No. 6,300,063 to Lipshutz, et al. 2001, which is fully incorporatedherein by reference, Genetic Bit Analysis (GBA™) which is described byGoelet, P. et al. (PCT Appl. No. 92/15712), peptide nucleic acid (PNA,Ren B, et al., 2004. Nucleic Acids Res. 32: e42) and locked nucleicacids (LNA, Latorra D, et al., 2003. Hum. Mutat. 22: 79-85) probes,Molecular Beacons (Abravaya K, et al., 2003. Clin Chem Lab Med. 41:468-74), intercalating dye [Germer, S. and Higuchi, R. Single-tubegenotyping without oligonucleotide probes. Genome Res. 9:72-78 (1999)],FRET primers (Solinas A et al., 2001. Nucleic Acids Res. 29: E96),AlphaScreen (Beaudet L, et al., Genome Res. 2001, 11(4): 600-8),SNPstream (Bell Pa., et al., 2002. Biotechniques. Suppl.: 70-2, 74,76-7), Multiplex minisequencing (Curcio M, et al., 2002.Electrophoresis. 23: 1467-72), SnaPshot (Turner D, et al., 2002. HumImmunol. 63: 508-13), MassEXTEND (Cashman J R, et al., 2001. Drug MetabDispos. 29: 1629-37), GOOD assay (Sauer S, and Gut I G. 2003. RapidCommun. Mass. Spectrom. 17: 1265-72), Microarray minisequencing(Liljedahl U, et al., 2003. Pharmacogenetics. 13: 7-17), arrayed primerextension (APEX) (Tonisson N, et al., 2000. Clin. Chem. Lab. Med. 38:165-70), Microarray primer extension (O'Meara D, et al., 2002. NucleicAcids Res. 30: e75), Tag arrays (Fan J B, et al., 2000. Genome Res. 10:853-60), Template-directed incorporation (TDI) (Akula N, et al., 2002.Biotechniques. 32: 1072-8), fluorescence polarization (Hsu T M, et al.,2001. Biotechniques. 31: 560, 562, 564-8), Colorimetric oligonucleotideligation assay (OLA, Nickerson D A, et al., 1990. Proc. Natl. Acad. Sci.USA. 87: 8923-7), Sequence-coded OLA (Gasparini P, et al., 1999. J. Med.Screen. 6: 67-9), Microarray ligation, Ligase chain reaction, Padlockprobes, Rolling circle amplification, Invader assay (reviewed in Shi MM. 2001. Enabling large-scale pharmacogenetic studies by high-throughputmutation detection and genotyping technologies. Clin Chem. 47: 164-72),coded microspheres (Rao K V et al., 2003. Nucleic Acids Res. 31: e66)and MassArray (Leushner J, Chiu N H, 2000. Mol Diagn. 5: 341-80).

It will be appreciated that nucleic acid substitutions can be alsoidentified in mRNA molecules derived from the stem cell or stem cellline of the present invention. Such mRNA molecules are first subjectedto an RT-PCR reaction following which they are either directly sequencedor be subjected to any of the SNP detection methods describedhereinabove.

The disease-causing mutations of the present invention can be present inthe stem cell or stem cell line of the present invention in aheterozygous (i.e., the presence of only one disease-causing mutation),homozygous (i.e., the presence of two identical disease-causingmutations), or double heterozygous (i.e., the presence of two differentdisease-causing mutations) form. It will be appreciated that the mode ofinheritance of the disease-causing mutation (i.e., dominant, recessive,co-dominant and/or imprinting) can affect the outcome of the mutation,i.e., the presence or absence of the alteration of the phenotype of thestem cell or stem cell line of the present invention.

Thus, while in the case of a dominant disorder (e.g., Myotonicdystrophy) stem cell or stem cell line which are heterozygote for adisease-causing mutation exhibit the alteration of the phenotype, in thecase of a recessive disorder, only stem cells or stem cell line whichare homozygous or double-heterozygous to disease-causing mutationsexhibit the alteration of the phenotype.

As is shown in Example 1 of the Examples section which follows, thepresent inventors have isolated the I-5 ES cell line which carries thePAX3-del28 (510del28 in SEQ ID NO:34) in a heterozygous form and whichis associated with van Waardenburg syndrome; the I-7 ES cell line whichcarries more than 50 repeats of the CTG trinucleotide as set forth inSEQ ID NO:22 in a heterozygous form and which is associated withMyotonic dystrophy; the I-8. and I-9 which carry the 1505C→T (P377L)mutation as set forth in SEQ ID NO:21 in a heterozygout form and whichis associated with metachromatic leukodystrophy and the J-3 ES cell linewhich carries the W1282X mutation as set forth in SEQ ID NO:24 in aheterozygous form and which is associated with cystic fibrosis.

As used herein, the phrase “alteration of the phenotype” refers tochanges in the shape and function of the cells including, but notlimited to changes in receptor binding, cell secretion, intracellularreactions which lead to upregulation or downregulation of certain genes,changes in the size and shape of the cells and/or the cellularcompartments (e.g., nucleus, cytoplasm, nucleolus), changes inproliferation and/or differentiation processes of the cells, and thelike. More specifically, the alteration of the phenotype of the presentinvention can be lysosomal accumulation of sulfatides in Schwann cells,periaxonal Schwann cells, macrophages, and spiral and vestibularganglion cell perikarya due to mutations causing metachromaticleukodystrophy (Coenen R, et al., cta Neuropathol (Berl). 2001; 101:491-8); defects in cAMP-activated whole-cell currents and Cl— transportin cell lines carrying cystic fibrosis mutations (Zamecnik P C et al.,Proc Natl Acad Sci U S A. 2004; 101: 8150-5); and defects in migrationand differentiation in muscle and neuronal cells carrying Myotonicdystrophy mutations (Yanowitz J L et al., Dev Biol. August 200415;272(2):389-402).

It will be appreciated that such alterations in the phenotype can bedetected using histological stains (May-Grünwald-Giemsa stain, Giemsastain, Papanicolau stain, Hematoxyline stain and/or DAPI stain), flowcytometry analysis of membrane bound markers using, e.g., afluorescence-activated cell sorting (FACS), biochemical assays (e.g.,using enzymatic assays), immunological assays (e.g., using specificantibodies), and/or RNA assays (e.g., using RT-PCR, Northern blot, RNAin situ hybridization and in situ RT-PCR), cell proliferation assays[e.g., using a MTT-based cell proliferation assay (Hayon, T. et al.,2003. Leuk Lymphoma. 44: 1957-62)], cell differentiation assays (Kohler,T., et al., 2000. Stem Cells.18: 139-47), apoptosis assays [e.g., usingthe Ethidium homodimer-1 (Molecular Probes, Inc., Eugene, Oreg., USA),the Tunnel assay (Roche, Basel, Switzerland), the live/deadviability/cytotoxicity two-color fluorescence assay (L-3224, MolecularProbes)], flow cytometry analysis [Lodish, H. et al., “Molecular CellBiology”, W. H. Freeman (Ed.), 2000], and the like.

In order to generate the isolated stem cell or stem cell line of thepresent invention, a single stem cell which carry a disease-causingmutation is isolated as described hereinabove from a human embryocarrying a disease-causing mutation (e.g., van Waardenburg syndrome,Myotonic dystrophy) and preferably cultured. Such a human embryo can bean embryo (at the blastocyst stage) which was subjected topre-implantation genetic diagnosis (POD) and was found to carrydisease-causing mutations. Methods of culturing ES cells are known inthe arts. Briefly, stem cells are plated on a matrix (e.g., Matrigel®™)or feeder cell layers (e.g., MEFs, foreskin feeder cells) in a celldensity which promotes cell survival and proliferation but limitsdifferentiation. Typically, a plating density of between about 15,000cells/cm² and about 200,000 cells/cm² is used.

It will be appreciated that although single-cell suspensions of stemcells are usually seeded, small clusters may also be used. To this end,enzymatic digestion utilized for cluster disruption (see Example 1 ofthe Examples section which follows) is terminated before stem cellsbecome completely dispersed and the cells are triturated with a pipettesuch that clumps (i.e., 10-200 cells) are formed. However, measures aretaken to avoid large clusters which cause cell differentiation.

According to preferred embodiments of the present invention, the culturemedium includes cytokines and growth factors needed for cellproliferation [e.g., basic fibroblast growth factor (bFGF) and leukemiainhibitor factor (LIF)], and factors such as transforming growth factorβ₁ (TGFβ₁) which inhibit stem cell differentiation.

Such a culture medium can be a synthetic tissue culture medium such asKo-DMEM (Gibco-Invitrogen Corporation products, Grand Island, N.Y., USA)supplemented with serum, serum replacement and/or growth factors.

Serum can be of any source including fetal bovine serum (FBS), definedFBS (HyClone, Utah, USA), goat serum, human serum and/or serumreplacement™ (Gibco-Invitrogen Corporation, Grand Island, N.Y. USA).

Culture medium, serum, and serum replacement can be obtained from anycommercial supplier of tissue culture products, examples includeGibco-Invitrogen Corporation (Grand Island, N.Y. USA), Sigma (St. LouisMo., USA), HyClone (Utah, USA) and the ATCC (Manassas, Va. USA).

The serum or serum replacement used by the present invention areprovided at a concentration range of 1% to 40%, more preferably, 5% to35%, most preferably, 10% to 30%.

Growth factors of the present invention can be used at any combinationand can be provided to the stem cells at any concentration suitable forES cell proliferation, while at the same time inhibit ES celldifferentiation.

As shown in Example 1 of the Examples section which follows, the EScells of the present invention which carry the disease-causing mutationswere cultured on MEFs in the presence of culture medium (80% KO-DMEM)supplemented with 20% defined FBS, 1 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% non-essential amino acid stocks and weremaintained in an undifferentiated state for at least 40 passages.

Alternatively, culturing the hES cells of the present invention can beeffected using a conditioned medium instead of serum or serumreplacement supplemented medium.

Conditioned medium is the growth medium of a monolayer cell culture(i.e., feeder cells) present following a certain culturing period. Theconditioned medium includes growth factors and cytokines secreted by themonolayer cells in the culture.

Conditioned medium can be collected from a variety of cells formingmonolayers in culture. Examples include MEF conditioned medium, foreskinconditioned medium, human embryonic fibroblasts conditioned medium,human fallopian epithelial cells conditioned medium, and the like.

Particularly suitable conditioned medium are those derived from humancells, such as foreskin-conditioned medium which is produced byculturing human foreskin cells in a growth medium under conditionssuitable for producing the conditioned medium.

Such a growth medium can be any medium suitable for culturing feedercells. The growth medium can be supplemented with nutritional factors,such as amino acids, (e.g., L-glutamine), anti-oxidants (e.g.,beta-mercaptoethanol) and growth factors, which benefit stem cell growthin an undifferentiated state. Serum and serum replacements are added ateffective concentration ranges as described elsewhere (U.S. patentapplication Ser. No. 10/368,045).

Feeder cells are cultured in the growth medium for sufficient time toallow adequate accumulation of secreted factors to support stem cellproliferation in an undifferentiated state. Typically, the medium isconditioned by culturing for 4-24 hours at 37° C. However, the culturingperiod can be scaled by assessing the effect of the conditioned mediumon stem cell growth and differentiation.

Selection of culture apparatus for conditioning the medium is based onthe scale and purpose of the conditioned medium. Large-scale productionpreferably involves the use of dedicated devices. Continuous cellculture systems are reviewed in Furey (2000) Genetic Eng. News 20:10.

Following accumulation of adequate factors in the medium, growth medium(i.e., conditioned medium) is separated from the feeder cells andcollected. It will be appreciated that the feeder cells can be usedrepeatedly to condition further batches of medium over additionalculture periods, provided that the cells retain their ability tocondition the medium.

Preferably, the conditioned medium is sterilized (e.g., filtration usinga 20 μM filter) prior to use. The conditioned medium of the presentinvention may be applied directly on stem cells or extracted toconcentrate the effective factor such as by salt filtration. For futureuse, conditioned medium is preferably stored frozen at −80° C.

During the culturing step the stem cells are monitored for theirdifferentiation state. Typically, undifferentiated stem cells have highnuclear/cytoplasmic ratios, prominent nucleoli and compact colonyformation with poorly discernable cell junctions.

As is shown in Example 1 of the Examples section which follows and inFIGS. 1 c-d, the present inventors have illustrated that the ES cells ofthe present invention which carry the disease-causing mutation displaycharacteristic morphology of undifferentiated ESCs, i.e., roundcolonies, clear borders, spaces between cells, high cytoplasm to nucleusratio and existence of two or four nucleoli.

Cell differentiation can be determined upon examination of cell ortissue-specific markers which are known to be indicative ofdifferentiation. Such tissue/cell specific markers can be detected usingimmunological techniques well known in the art [Thomson J A et al.,(1998). Science 282: 1145-7]. Examples include, but are not limited to,flow cytometry for membrane-bound markers, immunohistochemistry forextracellular and intracellular markers and enzymatic immunoassay, forsecreted molecular markers. Thus, primate ES cells may express thestage-specific embryonic antigen (SSEA) 4, the tumor-rejecting antigen(TRA)-1-60 and TRA-1-81.

As is shown in FIGS. 3 a-f in Example 1 of the Examples section whichfollows, ES cells carrying the Van Waardenburg disease-causing mutationof the present invention expressed the SSEA4, TRA-1-60 and TRA-1-81 cellsurface markers typical for undifferentiated cells.

Determination of ES cell differentiation can also be effected viameasurements of alkaline phosphatase activity. Undifferentiated human EScells have alkaline phosphatase activity which can be detected by fixingthe cells with 4% paraformaldehyde and developing with the Vector Redsubstrate kit according to manufacturer's instructions (VectorLaboratories, Burlingame, Calif., USA).

As is shown in Example 1 of the Examples section which follows, the I-5and I-7 stem cells which carry the WS1 and DM1 mutations, respectively,remained in an undifferentiated proliferation state for at least 41passages.

In addition to monitoring a differentiation state, stem cells are oftenalso being monitored for karyotype, in order to verify cytologicaleuploidity, wherein all chromosomes are present and not detectablyaltered during culturing. Cultured stem cells can be karyotyped using astandard Giemsa staining and compared to published karyotypes of thecorresponding species.

The stem cells of the present invention which carry disease-causingmutations of the WS1, DM1, CF and MLD genetic disorders retain a normalkaryotype i.e., 46, XX or 46, XY following at least 30 passages (seeExample 1 of the Examples section).

It will be appreciated that the stem cell or stem cell line of thepresent invention which carry the disease-causing mutation are likely topass the disease-causing mutation to any differentiated cell, tissue ororgan which is derived thereof.

As is shown in Example 2 of the Examples section which follows and inFIGS. 4 c-f, 5 and 6 a-d, the I-5 and I-7 ES cells were capable ofdifferentiating in vitro (embryoid bodies) and in vivo (teratomas) toall three embryonic germ layers, namely, ectoderm, mesoderm andendoderm. Such a pluripotent capacity was retained even following 40passages.

Thus, according to another aspect of the present invention there isprovided an isolated embryoid body comprising a plurality of cells atleast some of which carry a disease-causing mutation in a genomicpolynucleotide sequence thereof.

As used herein, the phrase “embryoid body” (EB) refers to morphologicalstructures comprised of a population of ES and/or EG cells which haveundergone differentiation. EBs formation initiates following the removalof differentiation blocking factors from ES cell cultures. In the firststep of EBs formation, ES cells proliferate into small masses of cellswhich then proceed with differentiation. In the first phase ofdifferentiation, following 1-4 days in culture for human ES cells, alayer of endodermal cells is formed on the outer layer of the smallmass, resulting in “simple EBs”. In the second phase, following 3-20days post-differentiation, “complex EBs” are formed. Complex EBs arecharacterized by extensive differentiation of ectodermal and mesodermalcells and derivative tissues.

The phrase “at least some” as used herein refers to a situation ofgenetic mosaicism in which the embryoid body was formed from a group ofstem cells part of which was carrying the disease-causing mutation ofthe present invention. According to preferred embodiments “at leastsome” refers to at least 1%, more preferably, at least 2%, morepreferably, at least 3%, at least 4%, 5%, 6%, 7%, 8%, 9%, 10,%, 11%,more preferably, between 12%-98%, more preferably, between 20%-80%, morepreferably, between 30-60%, most preferably, at least 50% of the cellscarry the disease-causing mutation of the present invention.

As is mentioned above, EBs are formed following the removal of ES cellsfrom feeder layer-, or matrix-based cultures into suspension cultures.ES cells removal can be effected using type IV Collagenase treatment fora limited time. Following dissociation from the culturing surface, thecells are transferred to tissue culture plates containing a culturemedium supplemented with serum and amino acids.

It will be appreciated that EBs can be collected at any time duringculturing and examined using an inverted light microscope. Thus, EBs canbe assessed for their size and shape at any point in the culturingperiod. Examples of various EBs structures are shown in FIGS. 4 a-b.

During the culturing step, EBs can be monitored for their viabilityusing methods known in the arts, including, but not limited to, DNA(Brunk, C. F. et al., Analytical Biochemistry 1979, 92: 497-500) andprotein (e.g., using the BCA Protein Assay kit, Pierce, TechnologyCorporation, New York, N.Y., USA) contents, medium metabolite indices,e.g., glucose consumption, lactic acid production, LDH (Cook J. A., andMitchell J. B. Analytical Biochemistry 1989, 179: 1-7) and mediumacidity, as well as by using the XTT method of detecting viable cells[Roehm, N. et al., J. Immunol. Meth. 142, 257-265 (1991); Scudierd, D.et al., Cancer Res. 48, 4827-4833 (1988); Weislow, O. et al., J. Natl.Cancer Inst. 81, 577-586 (1989)].

In addition, the viability of the EBs of the present invention can bealso assessed using various staining methods, including but not limitedto the fluorescent Ethidium homodimer-1 dye (excitation, 495 nm;emission, 635 nm) which is detectable in cells with compromisedmembranes, i.e., dead cells; the Tunnel assay which labels DNA breakscharacteristics of cells going through apoptosis; and the live/deadviability/cytotoxicity two-color fluorescence assay, available fromMolecular Probes (L-3224, Molecular Probes, Inc., Eugene, Oreg., USA).

The differentiation level of the EB cells can be monitored by followingthe loss of expression of Oct-4, and the increased expression level ofother markers such as α-fetoprotein, NF-68 kDa, α-cardiac and albumin.Methods useful for monitoring the expression level of specific genes arewell known in the art and include RT-PCR, RNA in situ hybridization,Western blot analysis and immunohistochemistry.

As is shown in FIGS. 4 c-f and 5, the EBs of the present invention whichcarry the WS1 or DM1 disease-causing mutations expressed neurofilament68 KD and nestin which represent the ectoderm layer, α-cardiac actin andtroponin which represent the mesoderm layer and albumin and insulinwhich represent the endoderm layer. In addition, the diminished Oct-4expression in 5-day-old EBs demonstrate the decrease in undifferentiatedES cells along with EB formation.

As is mentioned above, EBs are cultured in suspension cultures in thepresence of a culture medium suitable for EB differentiation.Preferably, such a culture medium also includes serum or serumreplacement, which are provided in a concentration of at least 10% or15%, respectively.

The EBs of the present invention can be at any age. Preferably, the EBsof the present invention are between 1-120 day-old, more preferablybetween 1-30 day-old, 1-10 day-old, more preferably, between 2-10day-old, most preferably, 5 day-old.

It will be appreciated that the stem cell, stem cell line or embryoidbody of the present invention can be further differentiate intodifferentiated cells, tissue or even organs.

Such differentiated cells, tissue or organs can be used to developdisease models of various genetic disorders. For example, osteoblastscarrying mutations in the OSF2/CBFA1 gene can be used to studycleidocranial dysplasia (CCD, Lee B et al., Nat Genet. 1997; 16:307-10); pancreatic cells carrying gain-of-function mutations in thecationic trypsinogen gene can be used to study hereditary pancreatitis(Tautermann G et al., Digestion. 2001; 64: 226-32); neuronal cellscarrying mutations in the TATA box-binding protein gene can be used tostudy spinocerebellar ataxia type 17 (Bruni A C et al., Arch Neurol.2004; 61: 1314-20); and mast cells carrying an activating mutation inc-kit which can be used to study mastocytosis (Dror Y et al., Br JHaematol. 2000; 108: 729-36).

Thus, according to another aspect of the present invention there isprovided an isolated differentiated cell, tissue or organ carrying atleast one disease-causing mutation in a genomic polynucleotide sequencethereof.

As used herein the phrase “differentiated cell” refers to any cell witha specialized function, shape and structure which can be derived fromthe stem cell, stem cell line or embryoid body of the present invention.Examples include, but are not limited to, neural cells, retina cells,epidermal cells, hepatocytes, pancreatic cells, osseous cells,cartilaginous cells, elastic cells, fibrous cells, myocytes, myocardialcells, bone marrow cells, endothelial cells, smooth muscle cells, andhematopoietic cells.

The phrase “tissue” refers to part of an organism consisting of anaggregate of cells having a similar structure and function. Examplesinclude, but are not limited to, brain tissue, retina, skin tissue,hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue,blood tissue, muscle tissue, cardiac tissue brain tissue, vasculartissue, renal tissue, pulmunary tissue, gonadal tissue, hematopoietictissue and fat tissue.

The phrase “organ” refers to a fully differentiated structural andfunctional unit in an animal that is specialized for some particularfunction. For example, head, brain, eye, leg, hand, heart, liver kidney,lung, pancreas, ovary, testis, and stomach.

The differentiated cell, tissue or organ of the present invention can beobtained by subjecting the stem cell, stem cell line or embryoid body todifferentiation conditions. Such conditions may include withdrawing oradding nutrients, growth factors or cytokines to the medium, changingthe oxygen pressure, or altering the substrate on the culture surface.

For example, embryonic stem cells can differentiate to osteoblasts(Bourne S. et al., Tissue Eng. 2004; 10: 796-806), hematopoietic cells(Kitajima K. Methods Enzymol. 2003; 365:72-83), vascular cells (Fraser ST., et al., Methods Enzymol. 2003; 365: 59-72), pancreatic precursors(Kahan B W et al., Diabetes. 2003; 52: 2016-24), neuronal precursors(Rathjen J, Rathjen P D. ScientificWorldJournal. March 2002 12; 2:690-700), astrocytes (Tang F, et al., Cell Mol Neurobiol. 2002; 22:95-101), and cardiac cells (Rolletschek A,. et al., 2004; Toxicol Lett.149: 361-9; Foley A, and Mercola M, 2004; Trends Cardiovasc Med. 14:121-5).

Following is a non-limiting description of a number of procedures andapproaches for inducing differentiation of EBs to lineage specificcells.

Neural Precursor Cells

To differentiate the EBs of the present invention into neuralprecursors, four-day-old EBs are cultured for 5-12 days in tissueculture dishes including DMEM/F-12 medium with 5 mg/ml insulin, 50 mg/mltransferrin, 30 nM selenium chloride, and 5 mg/ml fibronectin (ITSFnmedium, Okabe, S. et al., 1996, Mech. Dev. 59: 89-102). The resultantneural precursors can be further transplanted to generate neural cellsin vivo (Brüstle, O. et al., 1997. In vitro-generated neural precursorsparticipate in mammalian brain development. Proc. Natl. Acad. Sci. USA.94: 14809-14814). It will be appreciated that prior to theirtransplantation, the neural precursors are trypsinized and triturated tosingle-cell suspensions in the presence of 0.1% DNase.

Oligodendrocytes and Myelinate Cells

EBs of the present invention can differentiate to oligodendrocytes andmyelinate cells by culturing the cells in modified SATO medium, i.e.,DMEM with bovine serum albumin (BSA), pyruvate, progesterone,putrescine, thyroxine, triiodothryonine, insulin, transferrin, sodiumselenite, amino acids, neurotrophin 3, ciliary neurotrophic factor andHepes (Bottenstein, J. E. & Sato, G. H., 1979, Proc. Natl. Acad. Sci.USA 76, 514-517; Raff, M. C., Miller, R. H., & Noble, M., 1983, Nature303: 390-396]. Briefly, EBs are dissociated using 0.25% Trysin/EDTA (5min at 37° C.) and triturated to single cell suspensions. Suspendedcells are plated in flasks containing SATO medium supplemented with 5%equine serum and 5% fetal calf serum (FCS). Following 4 days in culture,the flasks are gently shaken to suspend loosely adhering cells(primarily oligodendrocytes), while astrocytes are remained adhering tothe flasks and further producing conditioned medium. Primaryoligodendrocytes are transferred to new flasks containing SATO mediumfor additional two days. Following a total of 6 days in culture,oligospheres are either partially dissociated and resuspended in SATOmedium for cell transplantation, or completely dissociated and a platedin an oligosphere-conditioned medium which is derived from the previousshaking step [Liu, S. et al., (2000). Embryonic stem cells differentiateinto oligodendrocytes and myelinate in culture and after spinal cordtransplantation. Proc. Natl. Acad. Sci. USA. 97: 6126-6131].

Mast Cells

For mast cell differentiation, two-week-old EBs of the present inventionare transferred to tissue culture dishes including DMEM mediumsupplemented with 10% FCS, 2 mM L-glutamine, 100 units/ml penicillin,100 mg/ml streptomycin, 20% (v/v) WEHI-3 cell-conditioned medium and 50ng/ml recombinant rat stem cell factor (rrSCF, Tsai, M. et al., 2000. Invivo immunological function of mast cells derived from embryonic stemcells: An approach for the rapid analysis of even embryonic lethalmutations in adult mice in vivo. Proc Natl Acad Sci USA. 97: 9186-9190).Cultures are expanded weekly by transferring the cells to new flasks andreplacing half of the culture medium.

Hemato-Lymphoid Cells

To generate hemato-lymphoid cells from the EBs of the present invention,2-3 days-old EBs are transferred to gas-permeable culture dishes in thepresence of 7.5% CO₂ and 5% O₂ using an incubator with adjustable oxygencontent. Following 15 days of differentiation, cells are harvested anddissociated by gentle digestion with Collagenase (0.1 unit/mg) andDispase (0.8 unit/mg), both are available from F.Hoffman-La Roche Ltd,Basel, Switzerland. CD45-positive cells are isolated using anti-CD45monoclonal antibody (mAb) M1/9.3.4.HL.2 and paramagnetic microbeads(Miltenyi) conjugated to goat anti-rat immunoglobulin as described inPotocnik, A. J. et al., (Immunology Hemato-lymphoid in vivoreconstitution potential of subpopulations derived from in vitrodifferentiated embryonic stem cells. Proc. Natl. Acad. Sci. USA. 1997,94: 10295-10300). The isolated CD45-positive cells can be furtherenriched using a single passage over a MACS column Miltenyi).

It will be appreciated that since EBs are complex structures,differentiation of EBs into specific differentiated cells, tissue ororgan may require isolation of lineage specific cells from the EBs.

Such isolation may be effected by sorting of cells of the EBs viafluorescence activated cell sorter (FACS) or mechanical separation ofcells, tissues and/or tissue-like structures contained within the EBs.

Methods of isolating EB-derived-differentiated cells via FACS analysisare known in the art. According to one method, EBs are disaggregatedusing a solution of Trypsin and EDTA (0.025% and 0.01%, respectively),washed with 5% fetal bovine serum (FBS) in phosphate buffered saline(PBS) and incubated for 30 min on ice with fluorescently-labeledantibodies directed against cell surface antigens characteristics to aspecific cell lineage. For example, endothelial cells are isolated byattaching an antibody directed against the platelet endothelial celladhesion molecule-1 (PECAM1) such as the fluorescently-labeled PECAM1antibodies (30884X) available from PharMingen (PharMingen, BectonDickinson Bio Sciences, San Jose, Calif., USA) as described inLevenberg, S. et al., (Endothelial cells derived from human embryonicstem cells. Proc. Natl. Acad. Sci. USA. 2002. 99: 4391-4396).Hematopoietic cells are isolated using fluorescently-labeled antibodiessuch as CD34-FITC, CD45-PE, CD31-PE, CD38-PE, CD90-FITC, CD117-PE,CD15-FITC, class I-FITC, all of which IgG1 are available fromPharMingen, CD133/1-PE (IgG1) (available from Miltenyi Biotec, Auburn,Calif.), and glycophorin A-PE (IgG1), available from Immunotech (Miami,Fla.). Live cells (i.e., without fixation) are analyzed on a FACScan(Becton Dickinson Bio Sciences) by using propidium iodide to excludedead cells with either the PC-LYSIS or the CELLQUEST software. It willbe appreciated that isolated cells can be further enriched usingmagnetically-labeled second antibodies and magnetic separation columns(WACS, Miltenyi) as described by Kaufman, D. S. et al., (Hematopoieticcolony-forming cells derived from human embryonic stem cells. Proc.Natl. Acad. Sci. USA. 2001, 98: 10116-10721).

An example for mechanical isolation of beating cardiomyocytes from EBsis disclosed in U.S. Pat. Appl. No. 20030022367 to Xu et al. Briefly,four-day-old EBs of the present invention are transferred togelatin-coated plates or chamber slides and are allowed to attach anddifferentiate. Spontaneously contracting cells, which are observed fromday 8 of differentiation, are mechanically separated and collected intoa 15-mL tube containing low-calcium medium or PBS. Cells are dissociatedusing Collagenase B digestion for 60-120 minutes at 37° C., depending onthe Collagenase activity. Dissociated cells are then resuspended in adifferentiation KB medium (85 mM KCl, 30 mM K₂HPO₄, 5 mM MgSO₄, 1 mMEGTA, 5 mM creatine, 20 mM glucose, 2 mM Na₂ATP, 5 mM pyruvate, and 20mM taurine, buffered to pH 7.2, Maltsev et al., Circ. Res. 75:233, 1994)and incubated at 37° C. for 15-30 min. Following dissociation cells areseeded into chamber slides and cultured in the differentiation medium togenerate single cardiomyocytes capable of beating.

It will be appreciated that the culturing conditions suitable for thedifferentiation and expansion of the isolated lineage specific cellsinclude various tissue culture medium, growth factors, antibiotic, aminoacids and the like and it is within the capability of one skilled in theart to determine which conditions should be applied in order to expandand differentiate particular cell types and/or cell lineages [reviewedin Fijnvandraat A C, et al., Cardiovasc Res. 2003; 58: 303-12;Sachinidis A, et al., Cardiovasc Res. 2003; 58: 278-91; Stavridis M Pand Smith A G, 2003; Biochem Soc Trans. 31(Pt 1): 45-9].

As is mentioned hereinabove, the differentiated stem cell line orembryoid body of the present invention which carry the disease-causingmutation can be used to identify agents suitable for treating suchgenetic diseases.

Thus, according to another aspect of the present invention there isprovided a method of identifying an agent suitable for treating adisorder associated with at least one disease-causing mutation.

As used herein “treating a disorder associated with at least onedisease-causing mutation” refers to treating an individual sufferingfrom a disorder such as a neurological disorder, a muscular disorder, acardiovascular disorder, an hematological disorder, a skin disorder, aliver disorder, and the like that is caused by the disease-causingmutation of the present invention.

The phrase “treating” refers to inhibiting or arresting the developmentof a disease, disorder or condition and/or causing the reduction,remission, or regression of a disease, disorder or condition in anindividual suffering from, or diagnosed with, the disease, disorder orcondition. Those of skill in the art will be aware of variousmethodologies and assays which can be used to assess the development ofa disease, disorder or condition, and similarly, various methodologiesand assays which can be used to assess the reduction, remission orregression of a disease, disorder or condition.

The method is effected by subjecting cells of the stem cell line or theembryoid body of the present invention to differentiating conditions tothereby obtain differentiated cells exhibiting an effect of the at leastone disease-causing mutation and exposing the differentiated cells to aplurality of molecules to identify at least one molecule (i.e., theagent) capable of regulating the effect of the at least onedisease-causing mutation on the differentiated cells.

As used herein, “exposing the differentiated cells” refers to subjectingthe differentiated cells of the present invention to various testmolecules.

The phrase “cells exhibiting an effect of the at least onedisease-causing mutation” refers to eukaryotic cells, preferablymammalian cells, more preferably, human cells, which include thedisease-causing mutation in a genomic polynucleotide sequence thereofand which phenotype (i.e., structure and function) is effected by thedisease-causing mutation. Such an effect can be a change in the size andshape of the cells and/or the cellular compartments (e.g., nucleus,cytoplasm, nucleolus), a change in receptor binding, cell secretion,intracellular reactions which lead to upregulation or downregulation ofcertain genes, a change in proliferation and/or differentiationprocesses of the cell, and the like.

Once the differentiated cells are obtained, the test molecules (e.g.,drugs, minerals, vitamins, and the like) are applied on thedifferentiated cells and the structure and function of the cell isdetected using the molecular, immunological and biochemical methodswhich are fully described hereinabove. Molecules which exert significantmodulations of the structure and/or function of the differentiated cellsbecome candidates for additional evaluations as suitable for treatingthe disorder associated with the disease-causing mutation of the presentinvention.

For example, to study the effect of abnormal repeat expansion of the CTGtrinucleotide of the DMPK on mental retardation associated with Myotonicdystrophy neuronal cells can be expanded from EBs which are generatedfrom the I-7 ES cell line (DM1) of the present invention. Briefly,four-day-old EBs are cultured under differentiating conditions [ITSFnmedium, Okabe, 1996 (Supra)] and the resultant neuronal precursors canbe tested for the activation of early (ERK1/2) and late (MAP2)differentiation markers, essentially as described in Quintero-Mora M L,et al. 2002; Biochem Biophys Res Commun. 295: 289-94.

To study the effect of a cystic fibrosis (CF) mutation on pancreasinsufficiency associated with CF, ES cells carrying a CF mutation (e.g.,N1303K) are subjected to pancreas precursor cell differentiation asdescribed in [Kahan B W, 2003 (Supra)]. Briefly, ES cells are removedfrom their feeder layer cultures using 2 mmol/l EDTA containing 2%chicken serum. Following 7 days in suspension cultures intact EBs areplated onto gelatin-coated surfaces at a density of 30-50 EBs per 13-mmglass coverslip and are allowed to further differentiate for 1-5 weeksin high-glucose DMEM containing 10% FCS. The resulting pancreasprecursors cells can be further compared to normal pancreas precursorcells with respect to gene expression patterns (e.g., insulin, glucagon,somatostatin, and pancreatic polypeptide) and cellular response tovarious drug molecules. For example, a drug molecule that will correctthe abnormality of the apical membrane of the proximal duct epithelialcells which results in dehydrated protein-rich secretions from theproximal duct epithelial cells (Nousia-Arvanitakis S. J ClinGastroenterol. 1999; 29: 138-42).

The effect of the disease-causing mutation on gene expression level canbe determined using methods known in the art. Following is anon-limiting list of RNA-based methods which can be used according tothe method of the present invention.

Northern Blot analysis: This method involves the detection of aparticular RNA in a mixture of RNAs. An RNA sample is denatured bytreatment with an agent (e.g., formaldehyde) that prevents hydrogenbonding between base pairs, ensuring that all the RNA molecules have anunfolded, linear conformation. The individual RNA molecules are thenseparated according to size by gel electrophoresis and transferred to anitrocellulose or a nylon-based membrane to which the denatured RNAsadhere. The membrane is then exposed to labeled DNA probes. Probes maybe labeled using radio-isotopes or enzyme linked nucleotides. Detectionmay be using autoradiography, colorimetric reaction orchemiluminescence. This method allows both quantitation of an amount ofparticular RNA molecules and determination of its identity by a relativeposition on the membrane which is indicative of a migration distance inthe gel during electrophoresis.

RT-PCR analysis: This method uses PCR amplification of relatively rareRNAs molecules. First, RNA molecules are purified from the cells andconverted into complementary DNA (cDNA) using a reverse transcriptaseenzyme (such as an MMLV-RT) and primers such as, oligo dT, randomhexamers or gene specific primers. Then by applying gene specificprimers and Taq DNA polymerase, a PCR amplification reaction is carriedout in a PCR machine. Those of skills in the art are capable ofselecting the length and sequence of the gene specific primers and thePCR conditions (i.e., annealing temperatures, number of cycles and thelike) which are suitable for detecting specific RNA molecules. It willbe appreciated that a semi-quantitative RT-PCR reaction can be employedby adjusting the number of PCR cycles and comparing the amplificationproduct to known controls.

RNA in situ hybridization stain: In this method DNA or RNA probes areattached to the RNA molecules present in the cells. Generally, the cellsare first fixed to microscopic slides to preserve the cellular structureand to prevent the RNA molecules from being degraded and then aresubjected to hybridization buffer containing the labeled probe. Thehybridization buffer includes reagents such as formamide and salts(e.g., sodium chloride and sodium citrate) which enable specifichybridization of the DNA or RNA probes with their target mRNA moleculesin situ while avoiding non-specific binding of probe. Those of skills inthe art are capable of adjusting the hybridization conditions (i.e.,temperature, concentration of salts and formamide and the like) tospecific probes and types of cells. Following hybridization, any unboundprobe is washed off and the slide is subjected to either a photographicemulsion which reveals signals generated using radio-labeled probes orto a colorimetric reaction which reveals signals generated usingenzyme-linked labeled probes.

In situ RT-PCR stain: This method is described in Nuovo G J, et al.[Intracellular localization of polymerase chain reaction (PCR)-amplifiedhepatitis C cDNA. Am J Surg Pathol. 1993, 17: 683-90] and Komminoth P,et al. [Evaluation of methods for hepatitis C virus detection inarchival liver biopsies. Comparison of histology, immunohistochemistry,in situ hybridization, reverse transcriptase polymerase chain reaction(RT-PCR) and in situ RT-PCR. Pathol Res Pract. 1994, 190: 1017-25].Briefly, the RT-PCR reaction is performed on fixed cells byincorporating labeled nucleotides to the PCR reaction. The reaction iscarried on using a specific in situ RT-PCR apparatus such as thelaser-capture microdissection PixCell I LCM system available fromArcturus Engineering (Mountainview, Calif.).

Oligonucleotide microarray—In this method oligonucleotide probes capableof specifically hybridizing with specific polynucleotide sequences areattached to a solid surface (e.g., a glass wafer). Each oligonucleotideprobe is of approximately 20-25 nucleic acids in length. To compare theexpression pattern of such polynucleotides in cells harboring adisease-causing mutation vs. control cells, RNA is preferably extractedfrom the cells, cell lines, embryoid bodies, tissue or organs of thepresent invention using methods known in the art (using e.g., a TRIZOLsolution, Gibco BRL, USA). Hybridization can take place using eitherlabeled oligonucleotide probes (e.g., 5′-biotinylated probes) or labeledfragments of complementary DNA (cDNA) or RNA (cRNA). Briefly, doublestranded cDNA is prepared from the RNA using reverse transcriptase (RT)(e.g., Superscript II RT), DNA ligase and DNA polymerase I, allaccording to manufacturer's instructions (Invitrogen Life Technologies,Frederick, Md., USA). To prepare labeled cRNA, the double stranded cDNAis subjected to an in vitro transcription reaction in the presence ofbiotinylated nucleotides using e.g., the BioArray High Yield RNATranscript Labeling Kit (Enzo, Diagnostics, Affymetix Santa ClaraCalif.). For efficient hybridization the labeled cRNA can be fragmentedby incubating the RNA in 40 mM Tris Acetate (pH 8.1), 100 mM potassiumacetate and 30 mM magnesium acetate for 35 minutes at 94° C. Followinghybridization, the microarray is washed and the hybridization signal isscanned using a confocal laser fluorescence scanner which measuresfluorescence intensity emitted by the labeled cRNA bound to the probearrays.

For example, in the Affymetrix microarray (Affymetrix®, Santa Clara,Calif.) each gene on the array is represented by a series of differentoligonucleotide probes, of which, each probe pair consists of a perfectmatch oligonucleotide and a mismatch oligonucleotide. While the perfectmatch probe has a sequence exactly complimentary to the particular gene,thus enabling the measurement of the level of expression of theparticular gene, the mismatch probe differs from the perfect match probeby a single base substitution at the center base position. Thehybridization signal is scanned using the Agilent scanner, and theMicroarray Suite software subtracts the non-specific signal resultingfrom the mismatch probe from the signal resulting from the perfect matchprobe.

Although cell profiling methods which analyze the transcriptome of thecells of the present invention are preferred for their accuracy and highthroughput capabilities, it will be appreciated that the presentinvention can also utilize protein analysis tools for profiling thecells of the cultures.

Expression and/or activity level of proteins expressed in the cells ofthe cultures of the present invention can be determined using methodsknown in the arts.

Enzyme linked immunosorbent assay (ELISA): This method involves fixationof a sample (e.g., fixed cells or a proteinaceous solution) containing aprotein substrate to a surface such as a well of a microtiter plate. Asubstrate specific antibody coupled to an enzyme is applied and allowedto bind to the substrate. Presence of the antibody is then detected andquantitated by a colorimetric reaction employing the enzyme coupled tothe antibody. Enzymes commonly employed in this method includehorseradish peroxidase and alkaline phosphatase. If well calibrated andwithin the linear range of response, the amount of substrate present inthe sample is proportional to the amount of color produced. A substratestandard is generally employed to improve quantitative accuracy.

Western blot: This method involves separation of a substrate from otherprotein by means of an acrylamide gel followed by transfer of thesubstrate to a membrane (e.g., nylon or PVDF). Presence of the substrateis then detected by antibodies specific to the substrate, which are inturn detected by antibody binding reagents. Antibody binding reagentsmay be, for example, protein A, or other antibodies. Antibody bindingreagents may be radiolabeled or enzyme linked as described hereinabove.Detection may be by autoradiography, calorimetric reaction orchemiluminescence. This method allows both quantitation of an amount ofsubstrate and determination of its identity by a relative position onthe membrane which is indicative of a migration distance in theacrylamide gel during electrophoresis.

Radio-immunoassay (RIA): In one version, this method involvesprecipitation of the desired protein (i.e., the substrate) with aspecific antibody and radiolabeled antibody binding protein (e.g.,protein A labeled with I¹²⁵) immobilized on a precipitable carrier suchas agarose beads. The number of counts in the precipitated pellet isproportional to the amount of substrate.

In an alternate version of the RIA, a labeled substrate and anunlabelled antibody binding protein are employed. A sample containing anunknown amount of substrate is added in varying amounts. The decrease inprecipitated counts from the labeled substrate is proportional to theamount of substrate in the added sample.

Fluorescence activated cell sorting (FACS): This method involvesdetection of a substrate in situ in cells by substrate specificantibodies. The substrate specific antibodies are linked tofluorophores. Detection is by means of a cell sorting machine whichreads the wavelength of light emitted from each cell as it passesthrough a light beam. This method may employ two or more antibodiessimultaneously.

Immunohistochemical analysis: This method involves detection of asubstrate in situ in fixed cells by substrate specific antibodies. Thesubstrate specific antibodies may be enzyme linked or linked tofluorophores. Detection is by microscopy and subjective or automaticevaluation. If enzyme linked antibodies are employed, a colorimetricreaction may be required. It will be appreciated thatimmunohistochemistry is often followed by counterstaining of the cellnuclei using for example Hematoxyline or Giemsa stain.

In situ activity assay: According to this method, a chromogenicsubstrate is applied on the cells containing an active enzyme and theenzyme catalyzes a reaction in which the substrate is decomposed toproduce a chromogenic product visible by a light or a fluorescentmicroscope.

In vitro activity assays: In these methods the activity of a particularenzyme is measured in a protein mixture extracted from the cells. Theactivity can be measured in a spectrophotometer well using colorimetricmethods or can be measured in a non-denaturing acrylamide gel (i.e.,activity gel). Following electrophoresis the gel is soaked in a solutioncontaining a substrate and colorimetric reagents. The resulting stainedband corresponds to the enzymatic activity of the protein of interest.If well calibrated and within the linear range of response, the amountof enzyme present in the sample is proportional to the amount of colorproduced. An enzyme standard is generally employed to improvequantitative accuracy.

It will be appreciated that large-scale proteomic analysis can be alsoemployed in order to identify biomarkers associated with thedisease-causing mutations of the present invention. For example, theproteins of the cells, cell lines, embryoid bodies, tissues or organs ofthe present invention can be subjected to various dissolving agents(e.g., SDS, Urea) followed by determination of protein sequencing ormass spectrometry analysis. Thus, the stem cell, stem cell line,embryoid body, differentiated cell, tissue or organ of the presentinvention which carry a disease-causing mutation can be used for drugdiscovery and testing, cell-based therapy, transplantation, productionof biomolecules, testing the toxicity and/or teratogenicity of compoundsand facilitating the study of developmental and other biologicalprocesses.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., Ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(Eds.) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., Ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., Ed. (1994); Stites et al.(Eds.), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (Eds.), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., Ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,Eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., Ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Generation of Human Embryonic Cell Lines Harboring GeneticMutations

Human ES cell lines were generated from discarded embryos blastocystsfollowing preimplantation genetic diagnosis (PGD) and the presence ofthe disease-causing-mutations in the ESCs was determined, as follows.

Materials and Experimental Methods

Blastocyst cultivation—In vitro fertilization was performed by sperminjection (ICSI) into oocytes retrieved following gonadotrophin-inducedovarian stimulation. Injected oocytes (18-19 hours post-ICSI) weremonitored for the presence of pronuclear formation and zygotes withnormal pronucleai were transferred (as drops under oil) for blastocystcultivation in the presence of the Cook growth medium [specialized Cookmedia for insemination (IM), growth (GM) and blastocyst development(BM), Queensland, Australia].

Seventy-six discarded embryos were donated by the PGD program at theRambam Medical Center; the donor couples signed consent forms which wereapproved by the hospital and national health committee. The donatedembryos were either embryos that underwent PGD with unclear resultswhose parents decided to not retrieve and/or with positiveidentification of disease-causing-mutations, or were found unsuitablefor embryo transfer according to the IVF grading.

Micromanipulation blastomere biopsy—Blastomeres having 6-8 cells on thethird day in culture were subjected to a blastomere biopsy, as follows.Each embryo was gently held by a holding micropipette (20 microndiameter aperture) and the zona pellucida was drilled using an aperturemicropipette (10-micron in diameter) filled with acid Tyrode's solution(pH 2.4; Sigma Chemical Co., St. Louis, Mo., USA). The resulting openingof the zona pellucida was slightly smaller than the size of theblastomere (˜40 microns). A 40-micron micropipette filled with PBS wasinserted through the opening, and the nearest blastomere(s) wasaspirated. For genetic analysis, each of the aspirated blastomere's cellwas transferred to a PCR tube.

Pre-implantation genetic diagnosis (PGD)—Prior to PCR amplification, theselected blastomere cell was lysed for one hour at 37° C. using 2 μl-of125 μg/ml PCR grade proteinase K (Roche Diagnostic GmbH, Mannheim,Germany) and 1 μA of 17 μM SDS (Sigma Chemical Co., St. Louis, Mo.,USA), prepared in nuclease free water (Promega, Madison Wis.). Theproteinase K reaction was stopped by heat inactivation (15 minutes at95° C.) and the PCR mixture was added directly to the cell lyzate. Thefirst PCR was performed by adding a 17 μl PCR reaction mixture to thecell lyzate and the nested PCR was performed by adding 2 μl of the firstPCR product into 18 μl of the nested PCR reaction mixture, to reach afinal volume of 20 μl in each case. PCR reactions included initialdenaturation for 5 minutes, followed by 35 cycles of denaturation (at95° C. for first PCR, or 94° C. for nested PCR), annealing (at the notedannealing temperature in Table 1, hereinbelow) and elongation (at 72°C.), for 30 seconds each, and a final elongation for 7 minutes at 72° C.PCR primers and conditions are listed in Table 1, hereinbelow. NestedPCR products were separated on a 3% nusieve agarose (BiowhittakerMolecular Applications, Rockland, Me. USA) and photographed under UVillumination. TABLE 1 PCR primers and conditions for genetic diagnosisDisorder Forward (F) and reverse (R) Composition of PCR Anneal. (Gene)primers (SEQ ID NO:) reaction mixture Temp. Myotonic First PCR: 1 IUBioTaq polymerase 65° C. Dystrophy F (101): and 1 × PCR buffer (DMPK)5′-CTTCCCAGGCCTGCAGTTTGCCCATC (Bioline), 10% DMSO, GenBank (SEQ ID NO:1)2 mM MgCl₂, 0.2 mM dNTP Accession No. R (102): and 2 pmole of each ofNM_004409 5′-GAACGGGGCTCGAAGGGTCCTTGTAGC the primers (SEQ ID NO:2)Nested PCR 1 IU Taq polymerse and 65° C. F (409): 1 × PCR buffer (Qiagen5′-GAAGGGTCCTTGTAGCCGGGAA GmbH, Hilden, Germany), (SEQ ID NO:3) 1.5 mmMgCl₂, 0.2 mM R (410): dNTP, Q-solution 5′-GGGATCACAGACCATTTCTTTCT(Qiagen) and 2 pmole of (SEQ ID NO:4) each of the PCR primers; Van FirstPCR 1 IU BioTaq polymerase 60° C. Waardenburg F: 5′-CTTCCCACAGTGTCCACTCCand 1 × PGR buffer syndrome (SEQ ID NO:5) (Bioline), 1.5 mM MgCl₂,(PAX3) R: 5′-GAGGATTGCAAGGCTTATGG 0.2 mM dNTP, 2 pmole of GenBank (SEQID NO:6) each of the PCR primers Accession No. Nested PCR 1 IU Taqpolymerse and 60° C. NM_000438 F: 5′-ACGGCAGGCCGCTGCCCAAC 1 × PCR buffer(Qiagen), (SEQ ID NO:7) 1.5 mM MgCl₂, 0.2 mM R: 5′-AGTCTGGGAGCCAGGAGdNTP, Q-solution (SEQ ID NO:8) (Qiagen) and 2 pmole of each of the PCRprimers Cystic F (w1): 1 IU Taq polymerse and 60° C. Fibrosis5′-TACCTATATGTCACAGAAGT 1 × PCR buffer (Qiagen (CFTR) R (w2): GmbH,Hilden, Germany), GenBank 5′-GTACAAGTATCAAATAGCAG 1.5 mM MgCl₂, 0.2 mMNo. dNTP, Q-solution M28668 (Qiagen) and 2 pmol of each of the PCRprimers Following PCR the fragment (270 bp long) is subjected torestriction enzyme analysis using the MnII restriction enzyme.metachromatic First PCR F (2098): 1 IU Taq polymerse and 60° C.leukodystrophy 5′-GCAGTCTCTCTTCTTCTAGC 1 × PCR buffer (Qiagen(Arylsulfatase R (2264): GmbH, Hilden, Germany), A) GenBank No.5′-AGGGGCCAGGGATCTAGGGC 1.5 mM MgCl₂, 0.2 mM AY271820 dNTP, Q-solution(Qiagen) and 2 pmole of each of the PCR primers Following PCR thefragment is subjected to restriction enzyme analysis using the AluIrestriction enzyme.

Derivation of hES cell lines—After digestion of the zona pellucida byTyrode's acidic solution (Sigma, St Louis, Mo., USA) or its mechanicalremoval, the exposed blastocysts were placed on mitotically inactivatedmouse embryonic fibroblast (MEF) feeder layers in the presence of aculture medium consisting of 80% KO-DMEM, 1 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% non-essential amino acid stock (all from GibcoInvitrogen corporation products, San Diego, Calif., USA products) andsupplemented with 20% defined FBS (HyClone, Utah, USA), Following 5-10days in culture, the intracellular mass (ICM) of the expanded blastocystwas excised (using a needle and a micropipettor) and transferred tofresh MEF covered plates. The pluripotent cells (derived from the ICM)were further cultured in the presence of the same culture medium andpassaged every 4-10 days, depending on the cell density.

Culture of hES cells—From passage 7-10 and onward, the cells werecultured on MEFs covered plates using a culture medium consisting of 85%KO-DMEM, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% non-essentialamino acid stock, 4 ng/ml basic fibroblast growth factor andsupplemented with 15% ko-serum replacement and were routinely passagedevery four to six days using 1 mg/ml type IV Collagenase (All productsfrom Gibco Invitrogen). For storage, the cells were frozen in liquidnitrogen using a freezing solution consisting of 10% DMSO (Sigma), 10%FBS (Hyclone) and 80% KO-DMEM.

PCR analysis of human ES cell lines—DNA was extracted from the ES celllines using the Genomic DNA isolation kit (Wizard, Promega, Madison,Wis., USA) according to the manufacturer's instructions and 2 μl ofgenomic DNA was employed for PCR analysis using the PCR primers andconditions listed in Table 1, hereinabove.

Karyotype analysis—Karyotype analysis was performed as previouslydescribed (Amit et al, 2003). ES cells metaphases were blocked usingcolcemid CaryoMax colcemid solution, Invitrogen, Grand island, N.Y.,USA) and nuclear membranes were lysed in an hypotonic solution accordingto standard protocols (International System for Human CytogeneticNomenclature, ISCN). G-banding of chromosomes was performed according tomanufacturer's instructions (Giemsa, Merck). Karyotypes of at least 20cells per sample were analyzed and reported according to the ISCN.

Immunohistochemistry—Human ES cells were fixed for 15 minutes in 4%paraformaldehyde, blocked for 20 minutes in 2% normal goat serum in PBSand incubated for overnight at 4° C. with 1:20 dilutions of SSEA1,SSEA3, SSEA4, TRA1-60, TRA1-81 mouse anti-human antibodies, provided byProf. P Andrews the University of Sheffield, England. Cells were thenwashed in PBS and further incubated with 1:100 dilutions of Donkeyanti-mouse IgG antibodies conjugated to the fluorochrome Cys 3 (ChemiconInternational, Temecula Calif., USA). Cells were visualized under aninverted fluorescent microscope (Inverted fluorescent microscope, CARLZeiss, Germany).

Experimental Results

Pre-implantation genetic diagnosis (PGD) identified blastocyst cellsharboring various disease-causing-mutations—To determine the presence orabsence of disease-causing-mutations of the Van Waardenburg (WS1),Myotonic Dystrophy (DM1), cystic fibrosis (CF) or metachromaticleukodystrophy (MLD), PGD was performed on single cell's DNA (derivedfrom a blastocyst) using PCR primers specific to the PAX3 (GenBankAccession No. NM_(—)000438), DMPK (GenBank Accession No. NM-004409),CFTR (GenBank Accession No. M28668), or Arylsulfatase A (GenBankAccession No. AY271820), respectively (data not shown).

Generation of ES cell lines from blastocysts—Out of the 76 discardedembryos, 31 were developed to the blastocyst stage. For ES cell linesisolation, the embryos were plated as a whole blastocyst on MEFs (FIG. 1a). Following 5-10 days in culture, the ICM outgrowth was detected in5/31 embryos (FIG. 1 b) and the pluripotent stem cells (isolated fromthe ICM) were transferred to MEF covered plates for further culturing.

Genetic analysis reveals the presence of the Van Waardenburg syndrome(WS) disease-causing-mutation in a human ES cell line—In order todetermine if cells of a human ES cell line which was derived from anIVF-blastocyst of a known Van Waardenburg family (family BU-53) carry aWS disease-causing-mutation, the DNA was subjected to PCR analysis usingthe PAX3-specific PCR primers (SEQ ID NOs:5-8). As is shown in FIG. 2 a,while DNA of a normal (i.e., unaffected) individual revealed a singleband of 100 bp, the DNA of the affected parent and the resultant humanES cell line, each exhibited two bands of 100 and 100-28 bp,corresponding to the wild-type allele and the 28 bp—deleted allele,respectively. Sequence analysis of the 100-28 allele confirmed thepresence of a 28 bp deletion at the 3′-end of exon 2 in the affectedparent and the 1-5 (WS1) ES cell line. The deletion sequence correspondsto nucleic acid coordinates 54129-54157 of GenBank Accession No.AC010980 which includes the genomic sequence of PAX3, to nucleic acidcoordinates 510-538 of GenBank Accession No. X15043 (SEQ ID NO:34) whichincludes part of the gene encoding PAX3, and in part (due to an exonboundary) to nucleic acid coordinates 662-682 of GenBank Accession No.NM_(—)000438 (SEQ ID NO:23) which includes the full length mRNA encodingPAX3.

Genetic analysis reveals the presence of the Myotonic Dystrophy(DM)—disease-causing-mutation in a human ES cell line—DNA extracted fromcells of a human ES cell line (I-7) which was derived from anIVF-blastocyst of a known DM family was subjected to PCR analysis usingthe DM specific primers (SEQ ID NOs:1-4). As is shown in FIG. 2 b, whenthe PCR products were electrophoresed (using an 8% polyacrylamide gel)and stained [using silver staining (Lerer I, et al., 1994, Am. J. Med.Gen. 52: 79-84)], abnormal expansions of the CTG repeats were observedin the DNA of the 1-7 (DM1) human ES cell line (1.4 and 3.0 Kb), as wellas in DNA of several DM-affected individuals.

Human ES cell lines harbor the cystic fibrosis or metachromaticleukodystrophy disease-causing-mutations—The J-3 or the I-8 and I-9 EScell lines were found to carry, in a heterozygous form, the W1282X orP377L (1505C→T in GenBank Accession No. NM_(—)000487, SEQ ID NO:21)genetic mutations which cause cystic fibrosis or metachromaticleukodystrophy (MLD), respectively (data not shown).

Human ES cells harboring genetic mutations exhibit normalcharacteristics of human ES cell lines—The I-7 (DM1) and I-5 (WS1) EScell lines harboring the myotonic dystrophy and Van Waardenburg syndromedisease-causing mutations, respectively, demonstrated colony and cellmorphology which are typical of human ES cell lines, i.e. round colonieswith clear borders, spaces between cells, high cytoplasm to nucleusratio and existence of two to four nucleoli (FIGS. 1 c-d). In addition,as is shown in FIGS. 3 a-f, immunohistochemistry staining of the I-5(WS1) ESCs using clonal primary antibodies for undifferentiated surfacemarkers revealed negative staining for stage-specific embryonic antigen(SSEA)-1, weak or no staining for SSEA3, and positive staining forSSEA4, tumor recognition antigen (TRA)-1-60 and TRA-1-81 as previouslyshown for human ES cell lines (Thomson at el, 1998; Reubinoff et al,2000). Similar results were obtained with the I-7 (DM1) ESCs following37 passages (not shown). Moreover, karyotype analysis which wasconducted on cells at passage 30 and 17 for the I-5 (SW1) and I-7 (DM1)cell lines, respectively, revealed a normal 46, XX karyptypes in atleast 40 cells in each case.

Thus, these results demonstrate for the first time, the generation ofhuman ES cell lines harboring disease-causing-mutations of the VanWaardenburg syndrome, Myotonic Dytrophy, cystic fibrosis ormetachromatic leukodystrophy. Such human ES cell lines can be used forstudying the molecular and physiological pathways leading to suchgenetic disorders and in developing suitable treatments for suchdisorders.

Example 2 Embryoid Bodies and Teratomas can be Generated from Human ESCell Lines Harboring Disease-Causing-Mutations

To further test the suitability of human ES cell lines harboringdisease-causing-mutations to differentiate into all three embryonic germlayers, ES cell lines were transferred to suspension culture or wereinjected into SCID mice, and the expression pattern of severaldifferentiation markers was determined in the resulting embryoid bodiesor teratomas, respectively.

Materials and Experimental Methods

Immunohistochemistry—was performed as described in Example 1,hereinabove.

EB formation—ES cells from four to six confluent wells (40-60 c²m) werecollected using 1 mg/ml type IV Collagenase (Invitrogen), further brokeninto small clumps using 1000 μl Gilson pipette tips, and cultured insuspension in 58-mm Petri dishes (Greiner, Germany). EBs were grown in80% KO-DMEM, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1%non-essential amino acid stock (all from Gibco Invitrogen) andsupplemented with 20% defined FBS (HyClone).

Teratoma formation—Cells from six confluent wells of a six-well plate(60 c²m) were harvested and injected into the rear leg muscle offour-week-old male SCID-beige mice (Harlan, Jerusalem Israel). Resultingteratomas were examined histologically, at least 12 weekspost-injection. Briefly, teratomas were fixed in 10% neutral-bufferedformalin, dehydrated in graduated alcohol (70%-100%) and embedded inparaffin. For histological examination, 1-5 μm sections weredeparafinized and stained with hematoxylin/eosin (H&E).

RT-PCR—Total RNA was isolated from either undifferentiated cells grownfor 34 and 41 passages post derivation, or from 10 day-old EBs usingTri-Reagent (Sigma, St. Louis, Mo.), according to the manufacturer'sprotocol. cDNA synthesis was performed from 1 μg total RNA using MMLVreverse transcriptase RNase H minus (Promega, Madison, Wis., USA). PCRreactions included an initial strand denaturation for 5 minutes at 94°C. followed by repeated cycles of denaturation (94° C. for 30 seconds),annealing at the noted temperatures (see Table 1, hereinbelow) for 30seconds and elongation at 72° C. for 30 seconds. PCR primers andreaction conditions used are described in Table 2, hereinbelow. PCRproducts were size-fractionated using 2% agarose gel electrophoresis.TABLE 2 RT-PCR primers and conditions for the identification ofembryonic germ layer specific markers Gene product (Accession Forward(F) and reverse (R) Reaction Size number) SEQ ID NOs. primers (5′→3′)Condition (bp) Oct-4 SEQ ID NO:9 F: GAGAACAATGAGAACCTTCAGGA 30 cycles219 (S81255) SEQ ID NO:10 R: TTCTGGCGCCGGTTACAGAACCA at 60° C. in 1.5 mMMgCl₂ Albumin SEQ ID NO:11 F: TGCTTGAATGTGCTGATGACAGGG 35 cycles 302(AF542069) SEQ ID NO:12 R: AAGGCAAGTCAGCAGCCATCTCAT at 60° C. in 1.5 mMMgCl₂ α-fetoprotein SEQ ID NO:13 F: GCTGGATTGTCTGCAGGATGGGGAA 30 cycles216 (BC027881) SEQ ID NO:14 R: TCCCCTGAAGAAAATTGGTTAAAAT at 60° C. in1.5 mM MgCl₂ NF-68KD SEQ ID NO:15 F: GAGTGAAATGGCACGATACCTA 30 cycles473 (AY1566990) SEQ ID NO:16 R: TTTCCTCTCCTTCTTCACCTTC at 60° C. in 2 mMMgCl₂ α-cardiac SEQ ID NO:17 F: GGAGTTATGGTGGGTATGGGTC 35 cycles 486actin SEQ ID NO:18 R: AGTGGTGACAAAGGAGTAGCCA at 65° C. (NM_005159) in 2mM MgCl₂ β-actin SEQ ID NO:19 F: ATCTGGCACCACACCTTCTACAATGAGCTGCG 35cycles 838 (NM_001101) SEQ ID NO:20 R: CGTCATACTCCTGCTTGCTGATCCACATCTGCat 62° C. in 1.5 mM MgCl₂

Experimental Results

ES cells harboring disease-causing-mutations spontaneously differentiateinto the three embryonic germ layer cell types in vitro—To verify thathuman ES cells harboring disease-causing-mutations are functionally, aswell as phenotypically consistent with normal human ES cells, ES cellwere removed from their feeder layers and were cultured in suspension.As is shown in FIGS. 4 a and b, both the I-7 (DM1) and the I-5 (WS1) EScell lines, respectively, spontaneously formed embryoid bodies (EBs)including cystic EBs.

The functionality of the isolated EBs was further tested by IHC usingvarious embryonic cell markers. As is further shown in FIGS. 4 c-f, EBsexpressed nestin which is derived from an ectodermal origin, insulin,which is from a endodermal origin, and troponin, a marker of themesodermal origin. These results demonstrate that the ES cell linesharboring disease-causing-mutations are capable of differentiating intoall three embryonic germ layers, i.e., mesoderm, endoderm and ectoderm.

ES-consistent gene expression within the EBs was further verified usingRT-PCR. As shown in FIG. 5, while undifferentiated cells expressed highlevels of Oct 4, a marker for pluripotent embryonic stem and germ cells(Pesce M, and Scholer H R., 2001, Stem Cells 19: 271-8), cells harvestedfrom five-day-old EBs expressed genes, which are associated withcellular differentiation including neurofilament (NF-68 kD) which isrelated with embryonal ectoderm, α-cardiac actin which is associatedwith embryonal mesoderm, and albumin which is associated with embryonalendoderm. The diminished Oct 4 expression in the EB sample obtained fromthe DM1 ES cell line was consistent with previous reports of diminishedOct 4 expression following differentiation of totipotent cells tosomatic lineages (Thomson J A, et al., 1998, Science 282: 1145-7;Reubinoff B E, et al., 2000, Nat. Biotechnol. 18: 399-404). As havepreviously reported elsewhere (Schuldiner M. et al., 2000, Proc NatlAcad Sci USA 97: 11307-12; Amit, M. et al., 2003, Biol. Reprod. 68:2150-2156; Kehat, I. et al., 2001, J Clin Invest 108: 407-14) ES cellcultures might have some degree of background differentiation. Indeed,some of the cell-specific genes, like α-fetoprotein, albumin anda-cardiac actin, were also expressed in the undifferentiated ES cells(FIG. 5, lanes 1 and 2).

Thus, these results demonstrate that human ES cells harboringdisease-causing-mutations are capable of creating functional EBsconsisting of all three embryonic germ layers.

Human ES cells harboring disease-causing-mutations differentiate intoembryonic germ layers in vivo—To further substantiate the ability ofhuman ES cells harboring disease-causing-mutations to differentiate intoembryonal germ layers, ES cells were tested for teratoma formation invivo. Following injection into the hindlimb muscle of SCID Beige mice,the I-7 (DM1) and I5 (WS1) ES cells were able to form teratomas. As isshown in FIGS. 6 a-d, each teratoma contained representative tissues ofthe three embryonic germ layers, including cartilage and muscle tissueof the mesodermal origin, gut-like epithelium of the endodermal origin,and nerve tissue which is of the ectodermal origin.

In conclusion, human ES cells harboring disease-causing-mutations suchas those causing myotonic dystrophy and Van Waardenburg syndromesexhibit phenotypic as well as functional characteristics of ES cellline. Following their differentiation in vitro (i.e., into EBs) and invivo (i.e., in teratomas), ES cells expressed genes associated with allthree embryonal germ layers.

Discussion

The pluripotency and immortality of hES cells may be utilized for thedevelopment of research models for genetic diseases such as DM and WS.The ability of ES cells to differentiate into any cell type of the adulthuman body can facilitate in understanding the processes affecting eachsystem. For example, directed differentiation of human ES cells carryingdisease-causing-mutations into cardiomyocytes and/or stratified muscle(for DM), or nerve and/or pigment producing cells (for WS), may proveinvaluable for understanding the pathogenesis of these diseases. Forsome of these differentiation models, directing protocols for human ESalready exist (Xu et al, 2002; Mummery et al, 2002; Reubinoff et al,2001; Zhang et al, 2001). Such differentiation models can be also usedfor in vitro drug testing.

In addition, the ES cell lines of the present invention can be used tomonitor the effect of the mutation during differentiation. For example,the role of PAX3 in early nerve development and the evolution of the(CTG)n repeats characterizing DM during continuous culturing of EScells.

Gene therapy is often based on targeted correction, using smallfragments of a corrected region of the gene (Colosimo et al, 2001). Theavailability of human ES cell lines harboring disease-causing-mutationssuch as the W1282X in the CFTR gene (causing cystic fibrosis) and theP377L (1505C→T in GenBank Accession No. NM_(—)000487 SEQ ID NO:21) inthe Arylsulfatase A gene (causing metachromatic leukodystrophy) wouldbenefit the development of targeted correction models for thesemutations.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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1-51. (canceled)
 52. An isolated stem cell or stem cell line carrying adisease-causing mutation in a genomic polynucleotide sequence thereof.53. The isolated stem cell or stem cell line of claim 52, wherein saidstem cell is of embryonic origin.
 54. The isolated stem cell or stemcell line of claim 52, wherein said stem cell is of human origin. 55.The isolated stem cell or stem cell line of claim 52, wherein saiddisease-causing mutation is selected from the group consisting of amissense mutation, a nonsense mutation, a frameshift mutation, areadthrough mutation, a promoter mutation, a regulatory mutation, adeletion, an insertion, an inversion, a splice mutation and aduplication.
 56. The isolated stem cell or stem cell line of claim 52,wherein said disease-causing mutation is selected from the groupconsisting of a missense mutation, a nonsense mutation, a frameshiftmutation, a readthrough mutation, a promoter mutation, a regulatorymutation, a deletion, an insertion, an inversion, a splice mutation anda duplication.
 57. The isolated stem cell or stem cell line of claim 52,wherein said disease-causing mutation is selected from the groupconsisting of the W1282X as set forth in SEQ ID NO:24 associated withcystic fibrosis, the PAX3-del28 (510del28 in SEQ ID NO:34) associatedwith van Waardenburg syndrome, more than 50 (CTG) repeats as set forthin SEQ ID NO:22 associated with Myotonic dystrophy and the 1505C→T(P377L) as set forth in SEQ ID NO:21 associated with metachromaticleukodystrophy.
 58. The isolated stem cell or stem cell line of claim52, wherein said stem cell is capable of being maintained in anundifferentiated state for at least 41 passages.
 59. The isolated stemcell or stem cell line of claim 52, wherein said stem cell exhibits akaryotype of 46, XX or 46, XY following at least 30 passages.
 60. Theisolated stem cell or stem cell line of claim 52, wherein said stem cellexhibits pluripotent capacity following 40 passages.
 61. An isolatedembryoid body comprising a plurality of cells at least some of whichcarry a disease-causing mutation in a genomic polynucleotide sequencethereof.
 62. The isolated embryoid body of claim 61, wherein saidembryoid body is derived from a stem cell or a stem cell line.
 63. Theisolated embryoid body of claim 62, wherein said stem cell is of humanorigin.
 64. The isolated stem cell or stem cell line of claim 62,wherein said stem cell exhibits a karyotype of 46, XX or 46, XYfollowing at least 30 passages.
 65. The isolated embryoid body of claim61, wherein said disease-causing mutation is selected from the groupconsisting of a missense mutation, a nonsense mutation, a frameshiftmutation, a readthrough mutation, a promoter mutation, a regulatorymutation, a deletion, an insertion, an inversion, a splice mutation anda duplication.
 66. The isolated embryoid body of claim 61, wherein saiddisease-causing mutation is associated with a genetic disorder selectedfrom the group consisting of cystic fibrosis (CF), myotonic dystrophy(DM), van Waardenburg syndrome (WS), metachromatic leukodystrophy (MLD),Gorlin disease, Huntington's disease (HD), spinal muscular atrophy (SMA)and Duchenne muscular dystrophy (DMD).
 67. The isolated embryoid body ofclaim 61, wherein said disease-causing mutation is selected from thegroup consisting of the W1282X as set forth in SEQ ID NO:24 associatedwith cystic fibrosis, the PAX3-del28 (510del28 in SEQ ID NO:34)associated with van Waardenburg syndrome, more than 50 (CTG) repeats asset forth in SEQ ID NO:22 associated with Myotonic dystrophy and the1505C→T (P377L) as set forth in SEQ ID NO:21 associated withmetachromatic leukodystrophy.
 68. The isolated embryoid body of claim61, wherein said embryoid body is capable of differentiating into cellsof the embryonic ectoderm, embryonic endoderm and/or embryonic mesoderm.69. An isolated differentiated cell, tissue or organ carrying at leastone disease-causing mutation in a genomic polynucleotide sequencethereof.
 70. The isolated differentiated cell, tissue or organ of claim69, wherein said differentiated cell, tissue or organ is of humanorigin.
 71. The isolated differentiated cell, tissue or organ of claim69, wherein said disease-causing mutation is selected from the groupconsisting of a missense mutation, a nonsense mutation, a frameshiftmutation, a readthrough mutation, a promoter mutation, a regulatorymutation, a deletion, an insertion, an inversion, a splice mutation anda duplication.
 72. The isolated differentiated cell, tissue or organ ofclaim 69, wherein said disease-causing mutation is associated with agenetic disorder selected from the group consisting of cystic fibrosis(CF), myotonic dystrophy (DM), van Waardenburg syndrome (WS),metachromatic leukodystrophy (MLD), Gorlin disease, Huntington's disease(HD), spinal muscular atrophy (SMA) and Duchenne muscular dystrophy(DMD).
 73. The isolated differentiated cell, tissue or organ of claim69, wherein said disease-causing mutation is selected from the groupconsisting of the W1282X as set forth in SEQ ID NO:24 associated withcystic fibrosis, the PAX3-del28 (510del28 in SEQ ID NO:34) associatedwith van Waardenburg syndrome, more than 50 (CTG) repeats as set forthin SEQ ID NO:22 associated with Myotonic dystrophy and the 1505C→T(P377L) as set forth in SEQ ID NO:21 associated with metachromaticleukodystrophy.
 74. A method of identifying an agent suitable fortreating a disorder associated with at least one disease-causingmutation, comprising: (a) generating a stem cell line or an embryoidbody carrying the at least one disease-causing mutation; (b) subjectingcells of said stem cell line or said embryoid body to differentiatingconditions to thereby obtain differentiated cells exhibiting an effectof the at least one disease-causing mutation and; (c) exposing saiddifferentiated cells to a plurality of molecules and identifying fromsaid plurality of molecules at least one molecule capable of regulatingsaid effect of the at least one disease-causing mutation on saiddifferentiated cells, said at least one molecule being the agentsuitable for treating the disorder associated with the at least onedisease-causing-mutation.
 75. The method of claim 74, wherein saidembryoid body is derived from a stem cell or a stem cell line.
 76. Themethod of claim 74, wherein said stem cell is of embryonic origin. 77.The method of claim 74, wherein said stem cell is of human origin. 78.The method of claim 74, wherein said stem cell exhibits a karyotype of46, XX or 46, XY following at least 30 passages.
 79. The method of claim74, wherein said disease-causing mutation is selected from the groupconsisting of a missense mutation, a nonsense mutation, a frameshiftmutation, a readthrough mutation, a promoter mutation, a regulatorymutation, a deletion, an insertion, an inversion, a splice mutation anda duplication.
 80. The method of claim 74, wherein said disease-causingmutation is associated with a genetic disorder selected from the groupconsisting of cystic fibrosis (CF), myotonic dystrophy (DM), vanWaardenburg syndrome (WS), metachromatic leukodystrophy (MLD), Gorlindisease, Huntington's disease (HD), spinal muscular atrophy (SMA) andDuchenne muscular dystrophy (DMD).
 81. The method of claim 74, whereinsaid disease-causing mutation is selected from the group consisting ofthe W1282X as set forth in SEQ ID NO:24 associated with cystic fibrosis,the PAX3-del28 (510del28 in SEQ ID NO:34) associated with vanWaardenburg syndrome, more than 50 (CTG) repeats as set forth in SEQ IDNO:22 associated with Myotonic dystrophy and the 1505C→T (P377L) as setforth in SEQ ID NO:21 associated with metachromatic leukodystrophy. 82.The method of claim 74, further comprising a step of isolating lineagespecific cells from said embryoid body prior to step (b).
 83. The methodof claim 82, wherein said isolating lineage specific cells is effectedby sorting of cells contained within said embryoid body via fluorescenceactivated cell sorter.
 84. The method of claim 82, wherein saidisolating lineage specific cells is effected by a mechanical separationof cells, tissues and/or tissue-like structures contained within saidembryoid body.